A Small Molecule Inhibitor of the MITF Molecular Pathway

Probe Report

MLPCN Probe Report Template V6.1

Version #: 2.0

Date submitted: March 29, 2013

Title: A Small Molecule Inhibitor of the MITF Molecular Pathway Authors: Patrick W. Faloon1, Melissa Bennion1, Warren S. Weiner2, Robert A. Smith2, Jacqueline Wurst1, Michel Weiwer1, Cathy Hartland1, Carrie M. Mosher1, Stephen Johnston1, Patrick Porubsky2, Benjamin Neuenswander2, Sivaraman Dandapani1, Benito Munoz1, Frank J. Schoenen2, Shailesh Metkar1, Rizwan Haq3, David E. Fisher3, Jeffrey Aubé2, Michelle Palmer1, Stuart L. Schreiber4,5 1Chemical Biology Platform, Broad Institute, 2University of Kansas Specialized Chemistry Center, 3Department of Dermatology, Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School 4Chemical Biology Program, Broad Institute, and 5Department of Chemistry and Chemical Biology, Harvard University Corresponding author email: [email protected] Assigned Assay Grant #: 1 R03 DA031089-01 Screening Center Name & PI: Broad Institute Probe Development Center, Stuart Schreiber Chemistry Center Name & PI: University of Kansas Specialized Chemistry Center, Jeffrey Aubé Assay Submitter & Institution: David E. Fisher, Department of Dermatology, Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School PubChem Summary Bioassay Identifier (AID): 488944 Abstract: Micropthalmia-associated transcription factor (MITF) is a lineage restricted basic helix-loop-helix

leucine zipper transcription factor that is essential for melanocyte development, function and

survival. 15% of human melanomas have MITF gene amplification (1). In addition, a vast

majority of melanomas are dependent upon MITF for survival. We set out to identify small

molecule inhibitors of MITF activity that would allow for better molecular characterization of

MITF’s role in melanoma. Using an MITF-dependent melanoma cell line, SK-MEL-5, in a cell-

based luminescence assay, we measured the promoter activity of a MITF target gene,

melastatin (TRPM-1), in a high throughput screen (HTS). 331,578 compounds from the NIH

MLPCN compound library were screened. Of these, 3,206 compounds were active (a hit rate of

0.96%). A chloronaphthoquinone (CID 1716436/SID 22416871) was identified in the primary

HTS as an inhibitor of TRPM-1 promoter activity. It had potent activity upon retesting in the

primary assay, as did several closely related analogs. Structure activity relationship (SAR)

studies were performed to improve potency and to minimize deleterious properties. These

efforts generated a probe (CID 12387471/ML329) with improved chemical properties and

selectivity. In particular, ML329 was not prone to nucleophilic glutathione addition, whereas the

initial hit underwent adduct formation. ML329 was tested in two MITF-dependent melanoma cell

viability assays, SK-MEL-5 and MALME-3M plus a MITF-independent cell line, A375. ML329

showed specific activity against the MITF-dependent cells, primary melanocytes but no effect on

the viability in A375 cells. ML329 reduced the expression of multiple MITF target genes,

including pigment-related genes and the cell cycle regulator CDK2. As a tool compound,

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ML329 will be useful in elucidating the role of MITF in melanocyte lineage development and in

melanoma disease progression.

Probe Structure & Characteristics:

CID/ML#

Target Name

IC50 (uM) [SID, AID]

Anti-target

IC50 (μM) [SID, AID]

Fold Selective

Secondary Assays: IC50 (uM) [SID, AID]

12387471/ML329

TRPM-1 promoter activity

1.2 uM [144221520,

651588]

A375 cytotoxicity

>35 [144221520,

651591] >30x

1) SK-MEL-5 cytotoxicity 0.75 uM

[144221520, 651586]

2) MALME-3M cytotoxicity 0.7 uM

[144221520, 651585]

ML329

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Recommendations for scientific use of the probe:

We have developed a small molecule probe (ML329) that inhibits the expression of numerous

micropthalmia-associated transcription factor (MITF) target genes and blocks the proliferation of

numerous cell lines that require MITF for proliferation. ML329 could directly or indirectly interact

with MITF or components of the MITF regulatory network. MITF is a major molecular node in the

development, proliferation and maintenance of melanocytes (2). It also has an important role in

the progression and persistence of melanomas. As a transcription factor that regulates cell

cycle and pigmentation, interference of MITF with ML329 will be useful in characterizing the

specific roles of MITF in melanoma and validate blockade of MITF function as a potential

treatment of melanoma. The probe will benefit many researchers investigating melanoma and

the underlying molecular changes in the early stages of oncogenesis and the subsequent

changes in disease progression and metastasis. An inhibitor of MITF will also benefit the study

of melanogenesis by parsing out MITF’s function in this biological process away from its role in

the development of other neural crest derived lineages, like the inner ear and osteoclasts (2,3).

MITF has been implicated in clear cell sarcoma and ML329 could be used to determine if it is

efficacious in that disease context (4).

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1 Introduction

The microphthalmia-associated transcription factor (MITF) was identified as the product of a

gene that affects murine coat color. Mice lacking all MITF function are devoid of pigment in fur

and in eyes due to a total absence of melanocytes (5). The MITF gene encodes a basic-helix-

loop-helix leucine zipper (bHLH-ZIP) transcription factor that homo- or heterodimerizes with the

related transcription factors: TFEB, TFE3 and TFEC. These transcription factors, collectively

termed the MiT family of transcription factors, are more ubiquitously expressed and unlike MITF

are not essential for melanocytic differentiation (6). However, all members of the MiT family bind

via their basic domains to identical DNA target sequences containing the canonical E-box

promoter element CACGTC or the non-palindromic sequence CACATG.

When its activity is up-regulated in normal melanocytes, MITF initiates a transcriptional program

leading to melanocyte differentiation, cell cycle arrest, survival and pigmentation. It directly

regulates the transcription of major pigmentation genes, including tyrosinase (7,8), tyrosinase-

related protein 1 gene (TYRP-1) (9), dopachrome tautomerase/tyrosinase-related protein-2

(DCT/TYRP-2), QNR-71 (10), silver (11), and AIM1 (12). It has also been suggested that MITF

may induce cell cycle arrest during melanocytic differentiation, potentially via transcriptional

targeting of the cyclic dependent kinase inhibitors p21, CDKN1A (13) and CDK4A (INK4A) (14).

The anti-apoptotic protein Bcl-2 is directly activated by MITF and supports the survival of

melanocytes since Bcl-2 knockout results in white coat-color due to melanocyte death (15).

A role for MITF in melanocyte survival is further supported by the consequence of MITF

mutation in mice and people: melanocyte death, rather than presence of unpigmented

melanocytes. Correspondingly, amplification and over-expression of MITF occurs in 15-20% of

melanomas, leading to its designation as a bona fide melanoma oncogene (1). Suppression of

MITF activity is lethal to melanomas, and high MITF expression is a poor prognostic factor in

melanoma patients, as MITF over-expression is associated with a decrease in 5-year overall

survival. Moreover, enforced MITF overexpression was shown to cooperate with the common

melanoma oncogene BRAF(V600E) to transform human melanocytes (1). These results

indicate that MITF can have either differentiative or tumorigenic effects depending on the

cellular context. Whereas physiologic activation of Bcl-2 expression may protect melanocytes

(for example, from ultraviolet light), its up-regulation in the context of melanoma may actually

contribute to this cancer’s notorious chemoresistance.

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The above results suggest that small molecule probes that suppress MITF would be useful not

only in understanding the biology of context-specific transcriptional control, but also for

developing therapeutic strategies for melanoma. With the exception of nuclear hormone

receptors, success in directly targeting transcription factors has been very limited (16).

Therefore, the identification of upstream druggable pathways that regulate MITF would be

important as an alternative therapeutic strategy.

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2 Materials and Methods

See subsections for a detailed description of the materials and methods used for each assay.

Materials and Reagents

Steady-Glo® Luciferase Assay System was purchased from Promega (Catalog No. E2550; Madison, WI) CellTiter-Glo® Luminescent Cell Viability Assay was purchased from Promega (Catalog No. G7573; Madison, WI) Cells to CT Bulk Lysis reagents purchased from Ambion (Catalog No. 4391851C; Grand Island, NY) Cells to CT Bulk RT reagents purchased from Ambion (Catalog No. 4391852C; Grand Island, NY) Light Cycler 480 Probes Master purchased from Roche (Catalog No. 4887301001) Human GAPD (GAPDH) Endogenous Control VIC/MGB probe/primer limited purchased from Applied Biosystems (Catalog No. 4326317E; Grand Island, NY) Human MITF FAM probe/primer set purchased from Applied Biosystems (Catalog No. 4331182 Hs01117294_m1; Grand Island, NY) Human TRPM1 probe/primer set purchased from Applied Biosystems (Catalog No. 4331182 Hs00170127_m1; Grand Island, NY) Human CDK2 probe/primer set purchased from Applied Biosystems (Catalog No. 4331182 Hs01548894_m1; Grand Island, NY) Human DCT probe/primer set purchased from Applied Biosystems (Catalog No. 4331182 Hs01098278_m1; Grand Island, NY) Human MLANA probe/primer set purchased from Applied Biosystems (Catalog No. 4331182 Hs00194133_m1; Grand Island, NY)

Cell Lines

The following cell lines were used in this study:

TRPM-1:luc is a SK-MEL-5 melanoma cell line that expresses firefly luciferase under the

control of the melastatin (TRPM-1) promoter. This cell line was used in the primary HTS

campaign and generated by the Fisher Lab.

SK-MEL-5 is the parental cell line to SKMEL5 TRPM-1:luc cells, and does not contain

the luciferase reporter. This cell line was obtained from ATCC (Catalog Number HTB-70;

Manassas, VA).

A375 obtained from ATCC (Catalog Number CRL-1619; Manassas, VA) is a melanoma

cell line characterized to be independent of MITF for growth and survival

MALME-3M was obtained from ATCC (Catalog Number HTB-64; Manassas, VA) is a

melanoma cell line dependent upon MITF activity for growth and survival.

2.1 Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided

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in Appendix A (Table A1). Refer to Appendix B for the detailed assay protocols.

2.1.1 SK-MEL-5 TRPM-1 Luciferase Reporter Assay (Primary Assay AID Nos. 493177, 493073, 493102, 540348, 624290, 624259, 624316, 624363, 624440, 624426, 624430, 651588, 651753)

The TRPM1 luciferase promoter construct was transfected into the SK-MEL-5 melanoma cell

line and a stable cell line was generated. This promoter is exquisitely sensitive to MITF over-

expression and suppression and contains three canonical E-box motifs within the cloned

promoter fragment (17). On day 0, cells were plated at 2,000 cells per well into white, opaque

384 well plates in phenol red-free media. On day 1, cells were treated with compounds or

positive control for 24 hours. On day 2, 20 uL of SteadyGlo (Promega) was added per well and

luminescence signal was determined with the Perkin-Elmer EnVision plate reader. Primary HTS

data were analyzed in Genedata Screener Assay Analyzer. All values were normalized against

DMSO treated samples and the positive control (18 uM parthenolide, CID 6473881). For the

HTS, the average of two replicates was used to rank order activity and to choose compounds

for retests. For dose studies, percent (%) activity was determined for each concentration and

the concentration response curves (CRCs) were generated with Genedata Screener’s

Condoseo.

2.1.2 SK-MEL-5 Cell Cytotoxicity Assay (SA 1: AID Nos. 493240, 540347, 624289, 624315, 624366, 624427, 624429, 624428, 651586)

SK-MEL-5 cells were treated with compounds for 24 hours, and then cell viability was measured

using the CellTiter-Glo Assay (Promega), a luciferase-based reagent that measures cellular

ATP levels. The compounds were tested at different concentrations to determine IC50 values.

Compounds that were active in the primary assay and toxic below 30 uM at 24 hours were

considered for probe development. Data were normalized against DMSO in Genedata

Screener’s Assay Analyzer. Curves were generated with Genedata Screener’s Condoseo and

showed percent (%) activity for the individual doses.

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2.1.3 A-375 Cell Cytotoxicity Assay (SA1: AID Nos. 540335, 540346, 624489, 624324,

624364, 624368, 624488, 624490, 624492, 651591)

A375 cells were treated with compounds for 24 hours, and then cell viability was measured

using the CellTiter-Glo Assay (Promega), a luciferase-based reagent that measures cellular

ATP levels. The compounds were tested at different concentrations to determine IC50 values.

Compounds that were active in the primary assay and were not toxic below 30 uM at 24 hours

were considered for probe development. Data were normalized against DMSO in Genedata



2.1.4 MALME-3M Cell Cytotoxicity Assay (SA1: AID Nos. 493191, 540339, 624299,

624362, 651584, 651585)

MALME-3M cells were treated with compounds for 24 hours, and then cell viability was

measured using the CellTiter-Glo Assay (Promega), a luciferase-based reagent that measures

cellular ATP levels. The compounds were tested at different concentrations to determine IC50

values. Compounds that were active in the primary assay and toxic below 30 uM at 24 hours

were considered for probe development. Data were normalized against DMSO in Genedata



2.1.5 qPCR Assay for MITF Expression (SAI: AID No. 651773)

SK-MEL-5 cells were treated with compounds for 24 hours. Next, cells were lysed with DNase I

(Ambion, from Cell to CT Lysis Mix). Lysed cells were delivered to a RT-PCR plate (Ambion,

Cells to CT RT Mix) and the plates were then processed for reverse transcription to create

cDNA. qPCR was performed by transferring cDNA from the RT-PCR plate to a qPCR plate

containing PCR master mix (Roche, Probes Master), FAM Taqman probe/primer set for the

target gene (human MITF, Applied Biosystem, 4331182 Hs01117294_m1), VIC Taqman

probe/primer set for a house keeping gene (human GAPDH, Applied Biosystems C10228) and

water. qPCR plates were cycled using a real-time PCR instrument (Roche, Light Cycler). Using

the instrument software, a cycle call was generated when each well enters log phase

amplification (Ct). The delta Ct value was determined by subtracting the Ct value of the control

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gene (GAPDH) from the Ct value of the target gene (MITF) in each well. The delta delta Ct

value of each compound treatment was determined by averaging the delta Ct values of the

mock well on each plate and subtracting that average from the delta Ct value of each compound

well. The compounds were tested at different concentrations to determine IC50 values. Data

were normalized against DMSO in Genedata Screener’s Assay Analyzer. Curves were

generated with Genedata Screener’s Condoseo and showed percent (%) activity for the

individual doses.

2.1.6 qPCR Assay for TRPM1 Expression (SAI: AID No. 651770)

Protocol is the same as 2.1.5 except the following primers and probes were used: FAM Taqman

probe/primer set for the target gene (human TRPM1, Applied Biosystem, 4331182

Hs00170127_m1), VIC Taqman probe/primer set for a house keeping gene (human GAPDH,

Applied Biosystems C10228)

2.1.7 qPCR Assay for CDK2 Expression (SAI: AID No. 651772)


probe/primer set for the target gene (human CDK2, Applied Biosystem, 4331182



2.1.8 qPCR Assay for DCT Expression (SAI: AID No. 651771)


probe/primer set for the target gene (human DCT, Applied Biosystem, 4331182



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2.1.9 qPCR Assay for MLANA Expression (SAI: AID No. 651795)


probe/primer set for the target gene (human MLANA, Applied Biosystem, 4331182



2.1.10 Cell proliferation assay of primary human melanocytes (SAI: AID No. 651920)

Primary human neonatal melanocytes were isolated from discarded foreskins by gentle dispase

treatment and grown in Ham’s F10 media supplemented with 7% FBS,

penicillin/streptomycin/glutamine, 0.1 mM 1-methyl-3-(2-methylpropyl)-7H-purine-2,6-dione

(IBMX), 50ng/mL 12-tetradecanoylphorbol 13-acetate (TPA), 1 µM Na3VO4 and 1 µM N(6),2'-O-

dibutyryladenosine 3':5' cyclic monophosphate (dbcAMP). Cells were plated at 4,000 cells per

well of a 384 well plate. On the following day, 10 nL of compound was added per well and

incubated for 24 hours. The compounds were tested at different concentrations to determine

IC50 values. At the end of compound treatment, cell viability was measured with CellTiter-Glo

(Promega) and luminescence measured with the PerkinElmer EnVision plate reader. Data were

normalized against DMSO in Genedata Screener’s Assay Analyzer. Curves were generated

with Genedata Screener’s Condoseo and showed percent (%) activity for the individual doses.

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2.2 Probe Chemical Characterization

The probe was prepared as described in Section 2.3 and Appendix C. 1H NMR, 13C NMR and

HRMS were used to characterize this compound and the results are consistent with the

proposed structure. UPLC purity of the material studied was 100% at 214 nm.

Summary of Known Probe Properties in PubChem

IUPAC Chemical Name 4-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)benzenesulfonamide

PubChem CID 12387471

Molecular Weight 328.34

Molecular Formula C16H12N2O4S

XLogP3-AA 1.4

H-Bond Donor 2

H-Bond Acceptor 6

Rotatable Bond Count 3

Exact Mass 328.051778

Topological Polar Surface Area 115

Figure 1. Stability of ML329 in PBS Buffer & GSH and DTT Stability Assays

MLS004556029 (CID 12387471, SID 144221520) was tested over a time course in a PBS stability assay (A), GSH

stability assay over 6 hours (B), DTT stability assay over 48 hours (C), and DTT stability assay for Ethacrynic Acid

over 8 hours (D) (for C and D, no DTT in blue and with 50 µM DTT in red). The percent of compound remaining in the

supernatant at the various time points is plotted.

C D

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2.3 Probe Preparation

O

O

1. CeCl3·7H2O; aq EtOH

2. sulfanilamide75 °C

O

O

HN

SNH2

O O

Scheme 1. Synthesis of the Probe (ML329)

The probe (ML329) was synthesized in one step from commercially available 1,4-

naphthoquinone and sulfanilamide using cerium(III) chloride heptahydrate as a Lewis acid

catalyst as shown in Scheme 1. The reaction was allowed to stir at 75 °C for three days

(unoptimized) then dilute citric acid was added to the reaction suspension and the insoluble

material was collected by filtration. The filter cake was washed with water, dried, then purified

by preparative RPLC.

Experimental procedures for the synthesis of the probe can be found in Appendix C.

2.4 Additional Analytical Analysis

ML329 was tested for stability in PBS/1% DMSO (Figure 1A), human serum and mouse serum.

ML329 was also tested for solubility and GSH/DTT adduct formation (Figures 1B/1C). All

results indicate that ML329 is very stable in PBS/1% DMSO, the GSH/DTT adduct assays, and

in the different serums. Experimental procedures for the analytical assays are provided in

Appendix D.

3 Results

Probe attributes:

Has an average IC50 value of 1.2 uM (+/- 0.1 uM) in the TRPM-1 reporter assay

Shows activity in two MITF-dependent melanoma cell lines (SK-MEL-5 & MALME-3M)

No apparent cytotoxicity in A375, a MITF-independent cell line (EC50= >35 uM).

Shows dose-dependent inhibition of the expression of multiple MITF target in multiple

qPCR assays (TRPM-1, MITF, CDK2, DCT, MLANA)

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To identify novel inhibitors of MITF, TRPM-1 promoter activity was measured with an

engineered cell line that allows for luminescence detection via the TRPM-1 promoter in a MITF-

dependent melanoma cell line, SK-MEL-5. The TRPM-1 promoter responds dynamically to

alterations of MITF function and is more sensitive than a standard cell cytotoxicity assay (17).

This primary cell-based assay was used in a pilot screen that identified parthenolide, the

positive control, as a compound that could decrease the luminescence signal. The assay was

optimized for automation and a high throughput screen was pursued. The automated assay

was first tested with a validation set of 2,240 compounds. These compounds were tested in

duplicate with in-plate neutral (DMSO) and positive controls (18 uM parthenolide). Robustness,

reproducibility, and variability parameters were analyzed before initiating the full HTS. The HTS

was run over the course of several weeks. Data were normalized relative to controls, and plate

patterns were corrected using a multiplicative algorithm in Genedata Screener Assay Analyzer.

For each compound, the average of the two replicates was determined and used for subsequent

analysis.

Determination of hits required several criteria: only assay plates with a Z’ greater than 0.5 were

accepted for analysis, compounds needed to reach 60% inhibition relative to 18 uM

parthenolide, and score in fewer than 10% of HTS assays listed in PubChem. In total, 331,578

compounds were screened. Of these, 3,206 compounds were considered active (a hit rate of

0.96%), 613 were inconclusive, and 315,055 were inactive. 1,064 compounds scored in over

10% of assays listed in Pubchem and were excluded from further study. Compounds were

clustered based upon chemical structure using a customized script in Pipeline Pilot. Clusters

were rated based upon structural liabilities and ranked accordingly. Substructures were

analyzed and compared to inactive compounds to identify inactive analogs. Representatives

from the more desirable clusters were selected for retest. In addition, a small number of inactive

analogs were chosen to provide initial structure/activity relationship (SAR) data during the retest

studies. Of these, 1,379 compounds were retested over a range of concentrations to validate

activity, and 583 compounds showed dose-dependent inhibition of TRPM-1 expression below

IC50 of 10 uM. 261 compounds were below IC50= 5 uM.

Since active compounds produce a decrease in signal in the primary assay, confirmation was

required that the reduction in luminescence was not a result of general cytotoxicity by the

compound or by inhibition of luciferase itself. Compounds were tested in several melanoma cell

lines, including A375, a human melanoma cell line that proliferates despite very low levels of

Page 14 of 124

MITF protein (17). In recent studies, siRNA-mediated knockdown of MITF does not alter A375

viability or growth rates and seems to persist independent of MITF regulation (4; Fisher lab,

unpublished data). In addition, compounds were tested for cytotoxicity in the parental cell line

SK-MEL-5 and another MITF dependent cell line, MALME-3M, to verify dose-dependent

inhibition of cell proliferation. Of the compounds that retested in the primary assay, 48

compounds showed the appropriate activity in the SK-MEL-5, MALME-3M and A375 assays.

All available dry powder samples of the remainder qualifying compounds were procured from

commercial sources and purity was determined. Only compounds over 90% pure were tested.

The compounds were retested in the primary assay and in the cytotoxicity assays. 11

compounds showed an IC50 value of less than 35 uM in A375 cells and were excluded from

further consideration. Approximately 44 compounds had an IC50 value below 10 uM in the

primary assay. Fifteen chemical scaffolds showed potencies below 10 uM, and three of these

were prioritized for follow-up assays. MITF is known to regulate the expression of a number of

lineage-specific melanogenesis genes and cell cycle regulators. Therefore, compounds were

screened for inhibition of gene expression in qPCR assays for the following genes: MITF,

TRPM-1, MLANA, BCL2A, and CDK2. Three scaffolds were put forward as candidates for

medicinal chemistry but only one scaffold met all of the assay cutoffs and was amenable to

analog production. The lead compound was CID 1716436 (Figure 2).

As of 01 November 2012, the original hit (SID 22416871, CID 1716436, MLS000680589, BRD-

K45681478-001-06-9) is listed as active in 48 of 617 PubChem assays (6 of which are part of

the MITF project). It registered an IC50 value in the primary assay of 4.1 uM (Figure 3A, AID

493177), and showed no cytotoxicity after 24 h in A375 cells (Figure 3B, AID 540335). In

addition, it showed potency close to the IC50 cutoff of 10 uM in the SK-MEL-5 and MALME-3M

cytotoxicity assays (Figure 3C and 3D, respectively). With its good potency and lack of

cytotoxicity in A375 cells, it was prioritized for further development. Analogs were designed and

synthesized, eventually leading to the more potent and more soluble probe, ML329 (see Section

3.4).

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Figure 2. The structure of MLS000680589 (CID 1716436)

MLS000680589

Figure 3. Dose response curves for initial hit, MLS000680589

MLS000680589 (SID 22416871, CID 1716436) was tested across a range of concentrations up to 35 uM in the

primary assay and several secondary assays. Concentration response curves were generated with Genedata

Screener Condeseo and show normalized percent activity for the individual doses. TRPM-1 promoter assay (AID

493177), IC50= 4.1 uM (A); A375 cytotoxicity assay (AID 540335), IC50>35 uM (B); SK-MEL-5 CellTiter-Glo (AID

493240), IC50= 14 uM (C) and MALME-3M CellTiter-Glo (AID 493191), IC50 = 15.6 uM (D). =replicate 1, Δ=replicate

2

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Table 1 Probe and Selected Analogs Assay Performance (Average IC50, uM)

CID / SID

Broad ID / KU ID

Structure

TRPM-1 promoter

assay

A375 CTG

SK-MEL-5 CTG

MALME-3M CTG

TRPM-1

qPCR

12387471 / 144221520

BRD-K73037408-001-01-5 / KUC111774N-03

1.2 70 0.1 0.7 0.15

81124 / 144221523

BRD-K29842115-001-07-4 / KUC111337N-02

5 60 2.7 3.4 0.6

72909 / 144221522

BRD-K75502546-001-01-9 / KUC111359N-02

3.2 64 8.5 7.3 2.4

279009 / 144221524

BRD-K48101260-001-01-8 / KUC111761N-02

6.6 70 1.2 13.5 IA

56951846 / 144221521

BRD-K93744531-001-01-2 / KUC111358N-02

7.7 70 14 18.6 6

56928031 / 135611193

BRD-K16604218-001-01-2 / KUC111109N

41.7 64 70 63 NT

NT= not tested, IA=inactive (IC50 >35 uM), Values in bold fail to meet probe criteria

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Figure 4. Critical Path for Probe Development

3.1 Summary of Screening Results

Refer to subsections for a detailed description of the results.

In the primary HTS, compounds were active if they decreased TRPM-1 expression as measured

by luminescence. The positive control, parthenolide (18 uM), caused a decrease in expression

that led to over a 6-fold reduction in signal. Compounds with greater than 60% activity of the

positive control were considered actives and chosen for confirmation studies (see Section 3.4

for details). Figure 4 displays the critical path for probe development. To explore SAR,

numerous analogs were synthesized and tested. Selected results are shown in Tables 2 to 8 in

Section 3.4.

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3.2 Dose Response Curves for Probe

Figure 5. Dose-dependent Activity of the Probe (ML329)

MLS004556029 (CID 12387471, SID 144221520) was tested across a range of concentrations up to 35 uM

in the primary assay and several secondary assays. Concentration response curves were generated with

Genedata Screener Condeseo and show normalized percent activity for the individual doses. TRPM-1

promoter assay (AID 651588), IC50= 1.2 uM (A); A375 cytotoxicity assay (AID 651591), IC50>35 uM (B); SK-

MEL-5 CellTiter-Glo (AID 651586), IC50= 0.75 uM (C) and MALME-3M CellTiter-Glo (AID 651585), IC50 =

0.88 uM (D). =replicate 1, Δ=replicate 2, ☐=replicate 3, ◊=replicate 4

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3.3 Scaffold/Moiety Chemical Liabilities

The solubility of the probe (ML329) was experimentally determined to be 0.4 µM in phosphate

buffered saline (PBS) solution (pH 7.4). The probe is stable in PBS buffer solution, with 100%

remaining after 48 hours (see Figure 1A). While the probe could be imagined to be reactive

toward a thiol nucleophile, the probe was found to be quite stable to glutathione (GSH), with

80% remaining after 6 hours (see Figure 1B), and dithiothreitol (DTT), with 100% remaining

after 48 hours (see Figure 1C). The probe is stable in human plasma with approximately 100%

remaining after a 5 hour incubation. See the Appendix D for the experimental procedures for

the solubility, PBS stability, GSH/DTT stability and plasma stability measurements.

3.4 SAR Tables

The naphthoquinone core was chosen based on potency and selectivity for MITF inhibition,

while remaining non-toxic to MITF independent melanocytes. Our focus was on improving the

biological activity and chemical stability through analog synthesis. To begin, a more detailed

analysis of the primary high throughput (AID 488899) and associated initial screens (AIDs

493073, 493191, 493240 and 540346) was performed (Table 2). Of the 341,348 compounds

reported as tested in AID 488899, there were 347 compounds that contained the benzoquinone

substructure without containing the 2-chlorobenzoquinone substructure. This list was further

culled by only allowing benzoquinones with a single fusion to another ring (173 compounds after

filter). A final structural filter was imposed where structurally complex and/or synthetically

challenging compounds were excluded to allow for efficient analog production. The last two

filters were based on compound biological promiscuity data: 140/154 compounds had PubChem

Active_AID_ratios of ≤ 15%, where the Active_AID_ratio is the ratio of the number of PubChem

bioassays where the compound tested active divided by the total number of bioassays in which

the compound was tested, as a rough surrogate for general bioactivity promiscuity; 37/140 had

a RankScore (a measure of activity) in AID 488899 of at least 60. All of these compounds

except one (SID 92764352; CID 386492; RankScore of 66 in AID 488899; not shown in Table

2) had a 1,4-naphthoquinone (or quinoline-5,8-dione) core.

Some of the compounds from the high throughput screen had been tested in the confirmatory

and secondary assays and these results are shown in Table 2. Therefore, analysis of the

compounds centered around the RankScore and confirmatory activities, where available. Since

there was not a strong correlation between these two numbers within the activity ranges of

Page 20 of 124

interest (≥60 RankScore and < 15 µM AC50), we used the structures in Table 2 to guide us in

forming a small matrix-type library including benzamide, sulfonamide, cyclic amine, and aniline

pharmacophores. A selection of these compounds was synthesized and tested, with some

additional compounds added to probe the structural requirements and tolerances of the system.

The biological assay data and physical properties of these analogs are presented in Tables 3-8.

Characterization data (1H NMR spectra and UPLC chromatograms) of these analogs are

provided in Appendix F.

In Table 3, direct chloro substitution on the naphthoquinone core (Table 3, entry 1) gave

desirable activity, but these analogs were found to react with glutathione and were removed

from further consideration as probe candidates. Maintaining the anilino groups (Table 3, entries

2-11) consistently provided active compounds, although the best anilino compounds were

appended with piperidine, piperazine, methoxy or hydrogen substituents (Table 3, entries 2-5).

Direct methyl substitution on the naphthoquinone core (Table 3, entry 15) attenuated target

potency, but removing the anilino functionality (Table 3, entry 13) maintained desirable potency

and selectivity. Appending the naphthoquinone with tertiary nitrogens (Table 3, entry 11) also

provided active compounds though potency was lost. Two potential probe candidates were

noted from this particular series (i.e., Table 3, entries 4 and 13), after the first round SAR

feedback.

Table 4 illustrates that replacing the anilino moiety with N-methylacetamides provided an

increase in potency, but at the expense of increasing toxicity shown by the antitarget activity

(Table 4, entries 1-6). N-methylacetamides were removed from further consideration due to

their associated toxicity.

Next, we evaluated a series of benzoyl substitutions on the naphthoquinone ring, and the results

are summarized in Table 5. Direct comparison of anilino compounds (e.g., Table 3, entries 2, 6,

and 9) with their benzoyl counterparts (e.g., Table 5, entries 1, 5, and 6) illustrates that the

benzoyl series gave comparable target potencies, while the antitarget potencies often

diminished below the advantageous absolute cut-off limits (e.g., Table 5, entries 1-3 and 5).

Appending the benzoyl moiety with electron-withdrawing groups resulted in similar activity

(Table 5, entry 2) as did extending the alkyl chain pendant to the piperazine ring (Table 5, entry

3). However, a phenyl substitution on the piperazine ring led to a significant loss in potency

(Table 5, entry 6; Table 3, entry 9).

Page 21 of 124

Table 6 contains data from benzene- and thiophene-substituted sulfonamide analogs. Halogen

substitutions improved the activities of thiophene analogs (Table 6, entry 3 and 4) but were

detrimental to other sulfonamides (Table 6, entry 1 and 2). Naphthoquinones with -H, -OMe,

and –NH2 substitutions proved equipotent to the piperazine analogs (Table 6, entries 5, 6, 8,

and 10) but piperidine substitution attenuated potency (Table 6, entry 7).

Appending electron-withdrawing groups to the anilino moiety, as shown in Table 7, gave a slight

increase in potency (Table 7, entries 1 and 2) compared to a slight loss of activity when

electron-donating groups were used (Table 7, entries 1 and 5). Potencies were improved further

by exchanging the N-methylpiperazines for N-ethyl- or N-isopropylpiperazines (e.g., Table 7,

entries 5-7). The piperidine and pyrrolidine substituents were generally more potent towards the

antitarget than the piperazines (Table 7, entries 8-11) and the morpholino-like analogs lost

target potency when compared in the SKMEL5 and MALME-3M assays (Table 7, entries 12 and

13). Anilino analogs presented in Table 7 had enhanced potencies over those presented in

Table 3, but still did not satisfy the probe criteria.

Table 8 shows the SAR results of the hydrogen-substituted naphthoquinones, which were

designed and synthesized based on the two potential probe candidates from the first round SAR

(Table 8, entries 1 and 3). Adding heteroatoms into the naphthoquinone ring potentiated the

antitarget potency (Table 8, entry 4). Ortho- and para-electron-withdrawing groups pendant to

the anilino moiety were well tolerated (Table 8, entries 6, 9, and 11) but meta- groups

attenuated potencies (Table 8, entries 7 and 10). para-Chloro substitution also attenuated target

potency (Table 8, entry 8). Extending the aromatic ring with a methylene linker was a significant

detriment to activity (compare Table 8, entries 8 and 12). Substitution at the para-position with a

sulfonamide resulted in the most potent compound that retained selectivity and therefore, was

nominated as the probe (Table 8, entry 9).

Page 22 of 124

Table 2. Nascent SAR derived from the HTS results

Entry CID / SID

Structure

R3 R4 R5 R6 Rank

Score TRPM1

(µM) SKMEL5

(µM)

MALME‐3M (µM)

A375

(µM) ActiveAIDRatio

(promiscuity)

R1 / R2

1 4112523 / 22405760

(4‐Chlorophenyl)amino / Benzamido H H H H 72 0.9 11.2 20.3 ND 6.8

2 2870836 / 17413023

Morpholino / N‐Methylacetamido H H H H 89 3.5 7.9 12.7 ND 12.6

3 3249350 / 49817142

4‐Methylpiperazin‐1‐yl / 4‐Acetamidophenylsulfonamido H H H H 83 3.6 22.8 8.9 ND 6.3

4 5163303 / 49817141

4‐Ethylpiperazin‐1‐yl / 4‐Methylphenylsulfonamido H H H H 70 4.2 21.9 11.9 36.6 6.2

5 937638 / 17511695

Methoxy / (4‐Hydroxyphenyl)amino H H H H 80 5.1 8.4 14.3 ND 7.3

6 5220114 / 49821145

4‐Ethylpiperazin‐1‐yl / 4‐Bromophenylsulfonamido H H H H 90 5.7 22.9 20.1 ND 7.6

7 3147878 / 14726375

Piperidin‐1‐yl / Phenylamino H H H H 75 6.7 12.7 18.2 ND 9.7

8 3763775 / 49817175

4‐Methylpiperazin‐1‐yl / Phenylsulfonamido H H H H 78 6.9 16.9 11.2 ND 5.3

9 3519010 / 26658687

4‐Methylpiperazin‐1‐yl / Thiophene‐2‐sulfonamido H H H H 77 7.0 ND 14.6 ND 2.5

10 1473358 / 17403066

Methyl / 2‐Oxo‐2‐phenylethyl H H H H 66 7.2 7.7 12.7 42.7 8.6

11 185478 / 22411775

Methyl / H OMe H H H 64 7.8 18.8 ND 37.8 8.0

12 793869 / 22414808

Methoxy / Phenyl H H H H 75 9.1 9.9 20.3 ND 6.7

13 4141238 / 17517418

m‐Tolylamino / Benzamido H H H H 61 10.0 19.7 25.3 34.0 8.8

14 3816676 / 49817427

4‐Carbamoylpiperidin‐1‐yl / 4‐Bromophenylsulfonamido H H H H 68 11.9 ND ND ND 4.2

15 4097208 / 22410291

(1,1‐Dioxidotetrahydrothiophen‐3‐yl)‐ (methyl)amino /

Acetamido H H H H 64 21.2 ND ND ND 6.2

Page 23 of 124

16 280616 / 92764020

Methoxy / Amino (N)* Me H H 113 ND ND ND ND 7.9

17 386227 / 92764347

(4‐Hydroxy‐3‐methoxyphenethyl)amino / H (N)* H H H 113 ND ND ND ND 8.6

18 3339005 / 14733361

Phenethylamino / N‐(4‐Fluorobenzyl)acetamido H H H H 104 ND ND ND ND 13.7

19 3342467 / 87337062

(1,1‐Dioxidotetrahydrothiophen‐3‐yl)‐ (methyl)amino /

Benzamido H H H H 103 ND ND ND ND 10.5

20 16871 / 85272353

Methoxy / H H H H H 102 ND ND ND ND 11.1

21 2914378 / 22403881

Propylamino / N‐(3‐Fluorophenyl)acetamido H H H H 98 ND ND ND ND 12.5

22 1405137 / 14741273

Morpholino / 1H‐Benzo[d][1,2,3]triazol‐1‐yl H H H H 98 ND ND ND ND 12.6

23 3793001 / 14729488

Benzylamino / N‐(4‐Fluorobenzyl)acetamido H H H H 98 ND ND ND ND 13.3

24 3743938 / 17401678

(3‐((4‐Nitrobenzoyl)oxy)phenyl)amino / 1H‐Benzo[d][1,2,3]triazol‐1‐yl H H H H 97 ND ND ND ND 5.1

25 376947 / 92763758

4‐(Pyridin‐2‐yl)piperazin‐1‐yl / H OH H H OH 92 ND ND ND ND 7.8

26 1951182 / 56422716


27 1423853 / 87346890


28 1422653 / 87346891


29 934233 / 26663804

Amino / Bromo H H H H 71 ND ND ND ND 7.9

30 3697347 / 49817138

4‐Ethylpiperazin‐1‐yl / Phenylsulfonamido H H H H 67 ND ND ND ND 5.8

31 5082182 / 17517457

(3‐Chloro‐4‐methylphenyl)amino / Benzamido H H H H 67 ND ND ND ND 9.0

32 247700 / 89855434

Phenylsulfonamido / H H H H H 66 ND ND ND ND 2.2

34 5182455 / 49647964

Amino / N‐Phenylacetamido H H H H 65 ND ND ND ND 5.2

35 3841172 / 17510130

Phenyl / Benzoyl H H H H 64 ND ND ND ND 11.4

Page 24 of 124

36 246348 / 89855329

Amino / Bromo H Me Me H 62 ND ND ND ND 2.2

37 284927 / 24828521

Acetamido / (2‐Methoxyphenyl)amino H H H H 61 ND ND ND ND 5.3

* Compounds marked as "(N)" have a quinoline-5,8-dione core. ND=not determined

Page 25 of 124

Table 3. Round 1 SAR and Anilino-substituted Naphthoquinone Compounds

SAR Analysis

Structure

Target Potency IC50 (µM) Antitarget Potency IC50 (µM)

Fold Selectivity

Entry CID / SID

Broad ID / KU ID * R1 R2 R3 n TRPM1 n SKMEL5 n MALME‐

3M n A375 A375 / TRPM1

1 1716436 / 134418986

BRD‐K45681478‐001‐11‐9 /

KUC110489N S ‐Cl

‐H 1 2.7 1 9.5 1 7.9 1 62.3 23

Solubility (PBS): ND µM Purity (UPLC): 100%

2 56951838 / 135659545

BRD‐K87235807‐001‐01‐6 /

KUC111332N S

‐Ph ‐H 1 6.1 1 23.9 1 16.4 1 45.7 7


3 3147878 / 135659539

BRD‐K15402285‐001‐07‐7 /

KUC111326N S

‐Ph ‐H 1 6.3 1 17.4 1 5.8 1 44.3 7


4 81124 /

135659550

BRD‐K29842115‐001‐07‐4 /

KUC111337N S ‐H ‐Ph ‐H 1 4.7 1 2.8 1 3.4 1 59.3 13

Solubility (PBS): 0.8 µM Purity (UPLC): 99%

5 931356 / 135724466

BRD‐K79227154‐001‐08‐7 /

KUC111409N S ‐OMe ‐Ph ‐H 1 6.3 1 3.2 1 8.2 1 59.0 9


6 3147877 / 135659538

BRD‐K22130095‐001‐02‐6 /

KUC111325N S

‐Ph ‐H 1 6.4 1 37.2 1 24.9 1 68.9 11


7 56951835 / 135659544

BRD‐K39913271‐001‐01‐4 /

KUC111331N S

‐Ph ‐H 1 9.8 1 62.4 1 70.0 1 69.8 7


Page 26 of 124

8 56951837 / 135659548

BRD‐K23298498‐001‐01‐5 /

KUC111335N S

‐Ph ‐H 1 10.8 1 70.0 1 70.0 1 64.6 6


9 56951849 / 135659549

BRD‐K83780064‐001‐01‐3 /

KUC111336N S

‐Ph ‐H 1 20.8 1 70.0 1 70.0 1 70.0 3


10 12230355 / 135724471

BRD‐K69430330‐001‐01‐1 /

KUC111414N S ‐Ph ‐Ph ‐H 1 11.3 1 30.0 1 41.9 1 70.0 6


11 4192149 / 136349438

BRD‐K93635281‐001‐02‐2 /

KUC111762N S ‐H ‐Ph ‐Me 1 16.2 1 2.3 1 ND 1 70.0 4


12 464135 / 135724468

BRD‐K12290434‐001‐01‐2 /

KUC111411N S ‐H ‐Me ‐H 1 12.6 1 61.8 1 44.9 1 70.0 6


13 72909 /

135659572

BRD‐K75502546‐001‐01‐9 /

KUC111359N S ‐H ‐H ‐H 1 3.2 1 8.5 1 7.3 1 64.0 20

Solubility (PBS): 100 µM Purity (UPLC): 92%

14 2755719 / 136349448

BRD‐K32884983‐001‐01‐3 /

KUC111772N S ‐H

‐H 1 70.0 1 53.3 1 ND 1 70.0 1


15 57339342 / 136349395

BRD‐K33808333‐001‐01‐8 /

KUC111719N S ‐Me

‐H 1 40.7 1 0.8 1 ND 1 70.0 2

Solubility (PBS): ND µM Purity (UPLC): 100% *P=purchased; S=synthesized

Page 27 of 124

Table 4. Round 1 SAR and N-methylacetamide-subsituted Naphthoquinone Compounds

SAR Analysis

Structure


Fold Selectivity

Entry CID / SID

Broad ID / KU ID * R n TRPM1 n SKMEL5 n MALME‐

3M n A375 A375 / TRPM1

1 56951830 / 135659553

BRD‐K05989866‐001‐01‐5 / KUC111340N S

1 0.4 1 4.4 1 2.0 1 14.4 36


2 56951839 / 135659554

BRD‐K41571295‐001‐01‐8 / KUC111341N S

1 0.4 1 4.9 1 2.5 1 11.3 27


3 2854727 / 135659552

BRD‐K66923876‐001‐01‐8 / KUC111339N S

1 0.7 1 4.6 1 4.9 1 29.0 40


4 2870836 / 135659551

BRD‐K62806031‐001‐10‐2 / KUC111338N S

1 2.1 1 7.8 1 7.4 1 26.3 12


5 56951834 / 135659560

BRD‐K61912369‐001‐01‐0 / KUC111347N S

1 0.1 1 0.6 1 0.7 1 11.0 119


6 56951845 / 135659555

BRD‐K31965646‐001‐01‐0 / KUC111342N S

1 0.4 1 4.4 1 3.1 1 4.8 11


Page 28 of 124

Table 5. Round 1 SAR and Benzoyl-substituted Naphthoquinone compounds

SAR Analysis

Structure


Fold Selectivity

Entry CID / SID

Broad ID / KU ID * R1 R2 n TRPM1 n SKMEL5 n MALME‐

3M n A375 A375 / TRPM1

1 56928026 / 135611200

BRD‐K22156309‐001‐01‐5 /KUC111116N S ‐H

1 0.8 1 8.5 1 3.6 1 29.8 37


2 56951842 / 135659563

BRD‐K04810559‐001‐01‐6 /KUC111350N S ‐Br

1 1.0 1 5.9 1 1.9 1 20.0 21


3 56928029 / 135611195

BRD‐K67283366‐001‐01‐3 /KUC111111N S ‐H

1 0.9 1 7.2 1 3.8 1 23.6 25


4 12408761 / 135659573

BRD‐K32454740‐001‐01‐9 /KUC111360N S ‐H ‐NH2 1 6.6 1 11.7 1 12.2 1 67.2 10


5 56928032 / 135611199

BRD‐K47317005‐001‐01‐1 /KUC111115N S ‐H

1 7.8 1 28.2 1 18.6 1 40.2 5


6 56928031 / 135611193

BRD‐K16604218‐001‐01‐2 /KUC111109N S ‐H

1 41.7 1 70.0 1 64.0 1 70.0 2


Page 29 of 124

Table 6. Round 1 SAR Benzene- and Thiophene-substituted Naphthoquinone Compounds

SAR Analysis

Structure


Fold Selectivity

Entry CID / SID

Broad ID / KU ID * R1 R2 n TRPM1 n SKMEL5 n MALME‐

3M n A375 A375 / TRPM1

1 3763775 / 135659557

BRD‐K27688565‐001‐07‐8 /KUC111344N S

‐Ph 1 0.9 1 14.6 1 7.3 1 37.5 42


2 3349693 / 135611189

BRD‐K07535132‐001‐01‐2 /KUC111105N S

1 3.4 1 45.1 1 70.0 1 60.5 18


3 3519010 / 135631293

BRD‐K77281287‐001‐07‐8 /KUC111280N S

1 2.6 1 42.2 1 11.7 1 65.8 25


4 56951831 / 135659568

BRD‐K05052689‐001‐01‐7 /KUC111355N S

1 3.0 1 28.7 1 9.8 1 59.8 20


5 247700 / 135724467

BRD‐K26127011‐001‐03‐7 /KUC111410N S ‐H ‐Ph 1 7.1 1 8.9 1 13.6 1 70.0 10


6 56973480 / 135724472

BRD‐K70479708‐001‐01‐9 /KUC111415N S ‐H

1 6.5 1 12.8 1 27.8 1 70.0 11


7 56951850 / 135659559

BRD‐K99619817‐001‐01‐5 /KUC111346N S

1 21.8 1 50.3 1 48.8 1 70.0 3


Page 30 of 124

8 56973487 / 135724462

BRD‐K39703137‐001‐01‐3 /KUC111405N S ‐NH2 ‐Ph 1 7.2 1 15.4 1 15.5 1 66.2 9


9 56928033 / 135611192

BRD‐K78795995‐001‐01‐2 /KUC111108N S

1 25.3 1 55.5 1 51.7 1 70.0 3


10 56951846 / 135659571

BRD‐K93744531‐001‐01‐2 /KUC111358N S ‐OMe ‐Ph 7.7 14.1 18.6 70.0 9

Solubility (PBS): 100 µM Purity (UPLC): 98% *P=purchased; S=synthesized

Page 31 of 124

Table 7. Round 2 SAR and Anilino- and Nitrogen-heterocycle-substituted Naphthoquinone Compounds

SAR Analysis

Structure


Fold Selectivity

Entry Previous Entry

CID / SID

Broad ID / KU ID * R1 R2 n TRPM1 n SKMEL5 n

MALME‐3M n A375 A375 /

TRPM1

1 Table 3 / Entry 2

56951838 / 135659545

BRD‐K87235807‐001‐01‐6 /

KUC111332N S ‐H

1 6.1 1 23.9 1 16.4 1 45.7 7


2 ‐ 57339347 /

136349422

BRD‐K57735325‐001‐01‐4 /

KUC111746N S 2,4‐diF

1 11.4 1 9.9 1 25.4 1 70.0 6


3 ‐ 57339336 / 136349421

BRD‐K75052540‐001‐01‐1 /


1 7.4 1 9.0 1 26.3 1 70.0 9


4 ‐ 57339354 /

136349423

BRD‐K56959705‐001‐01‐1 /


1 6.0 1 8.7 1 25.4 1 70.0 12


5 ‐ 57339334 /

136349398

BRD‐K50459480‐001‐01‐9 /

KUC111722N S 4‐OMe

1 24.9 1 16.4 1 ND 1 70.0 3


6 ‐ 57339364 /

136349397

BRD‐K23290885‐001‐01‐3 /


1 6.9 1 12.2 1 ND 1 70.0 10


7 ‐ 57339337 / 136349399

BRD‐K74109130‐001‐01‐6 /


1 3.3 1 16.6 1 ND 1 45.1 14

Page 32 of 124

8 Table 3 / Entry 3

3147878 / 135659539

BRD‐K15402285‐001‐07‐7 /

KUC111326N S ‐H

1 6.3 1 17.4 1 5.8 1 44.3 7


9 ‐ 57339349 /

136349420

BRD‐K97822234‐001‐01‐2 /


1 0.9 1 3.6 1 ND 1 21.8 23


10 ‐ 57339343 /

136349424

BRD‐K12371913‐001‐01‐0 /


1 0.7 1 2.3 1 ND 1 8.1 12


11 ‐ 1389332 / 136349396

BRD‐K75845864‐001‐01‐6 /


1 7.2 1 3.7 1 11.2 1 56.2 8


12 Table 3 / Entry 6

3147877 / 135659538

BRD‐K22130095‐001‐02‐6 /

KUC111325N S ‐H

1 6.4 1 37.2 1 24.9 1 68.9 11



56951835 / 135659544

BRD‐K39913271‐001‐01‐4 /

KUC111331N S ‐H

1 9.8 1 62.4 1 70.0 1 69.8 7



56951849 / 135659549

BRD‐K83780064‐001‐01‐3 /

KUC111336N S ‐H

1 20.8 1 70.0 1 70.0 1 70.0 3


Page 33 of 124

Table 8. Round 2 SAR and Hydrogen-substituted Naphthoquinone compounds

SAR Analysis

Structure


Fold Selectivity

Entry Previous Entry

CID / SID

Broad ID / KU ID * R1 R2 R3 X n TRPM1 n SKMEL5 n

MALME‐3M n A375 A375 /

TRPM1


72909 / 135659572

BRD‐K75502546‐001‐01‐9 /

KUC111359N S ‐H ‐H ‐H CH 1 3.2 1 8.5 1 7.3 1 64.0 20

Solubility (PBS): 100 µM Purity (UPLC): 92%


464135 / 135724468

BRD‐K12290434‐001‐01‐2 /

KUC111411N S ‐Me ‐H ‐H CH 1 12.6 1 61.8 1 44.9 1 70.0 6


3 Table 3 / Entry 4

81124 / 135659550

BRD‐K29842115‐001‐07‐4 /

KUC111337N S ‐Ph ‐H ‐H CH 1 4.7 1 2.8 1 3.4 1 59.3 13


4 ‐ 3976 /

136349454

BRD‐K62792802‐001‐04‐4 /

KUC111778N P ‐Ph ‐H ‐H N 1 0.4 1 0.2 1 ND 1 16.2 39



4192149 / 136349438

BRD‐K93635281‐001‐02‐2 /

KUC111762N S ‐Ph ‐Me ‐H CH 1 16.2 1 2.3 1 ND 1 70.0 4


6 ‐ 24861930 /

136349443

BRD‐K12528837‐001‐01‐8 /

KUC111767N S

‐H ‐H CH 1 5.4 1 0.2 1 ND 1 70.0 13


7 ‐ 57339353 /

136349444

BRD‐K04158422‐001‐01‐5 /

KUC111768N S

‐H ‐H CH 1 23.9 1 0.7 1 ND 1 22.3 1


Page 34 of 124


57339342 / 136349395

BRD‐K33808333‐001‐01‐8 /

KUC111719N S

‐H ‐Me CH 1 40.7 0.8 1 ND 1 70.0 2


9 ‐ 12387471 / 144221520

BRD‐K73037408‐001‐01‐5 /

KUC111774N‐03 S

‐H ‐H CH 1 1.2 1 0.1 1 0.7 1 70.0 58


10 ‐ 10515679 / 136349446

BRD‐K95690357‐001‐01‐6 /

KUC111770N S

‐H ‐H CH 1 18.9 1 0.7 1 ND 1 70.0 4


11 ‐ 279009 /

136349437

BRD‐K48101260‐001‐01‐8 /

KUC111761N S

‐H ‐H CH 1 6.6 1 1.2 1 13.5 1 70.0 11



2755719 / 136349448

BRD‐K32884983‐001‐01‐3 /

KUC111772N S

‐H ‐H CH 1 70.0 1 53.3 1 ND 1 70.0 1


13 ‐ 247700 /

135724467

BRD‐K26127011‐001‐03‐7 /

KUC111410N S

‐H ‐H CH 1 7.1 1 8.9 1 13.6 1 70.0 10



56973480 / 135724472

BRD‐K70479708‐001‐01‐9 /

KUC111415N S

‐H ‐H CH 1 6.5 1 12.8 1 27.8 1 70.0 11


Page 35 of 124

3.5 Cellular Activity

The primary assay and all of the secondary assays were cell-based, all lead compounds

showed activity in cells. In the medicinal chemistry phase of our studies, ML329 and its analogs

were tested in multiple cell-based experiments. ML329 consistently showed low micromolar

activity in multiple cell-based assays and has good permeability and solubility properties.

ML329 also showed potent activity in primary human melanocytes (IC50= 7 uM, AID 651920)

(Appendix I). The counterscreen with A375 cells helped to remove compounds that possessed

non-specific cellular toxicity.

3.6 Profiling Assays

ML329 was run in a panel of assays with PanLabs/Eurofins and showed little to no inhibition

with the exception of human Adenosine A2A where it showed 50% inhibition at 10 uM. See

Appendix H for more details.

4 Discussion

Figure 6. A visual summary of SAR performed for ML329

The aim of this project was to identify suppressors of MITF activity via a robust and sensitive

primary screen. We identified ML329, a potent probe that inhibits the growth of multiple MITF-

dependent cell lines, reduces the expression of MITF and several MITF-regulated genes.

ML329 meets all of the probe attributes and these are listed in Table 9. The long-term goal is to

use these probes in elucidating the molecular pathways regulating MITF activity with the goal of

translating this knowledge into therapeutic opportunities.

1. Mono‐ and multi‐substituted anilines2. N‐Aryl sulfonamides

3. N‐Acyl groups, e.g., N‐Methylacetamides and benzoylamides

(Sulfonamides and anilines provided optimal potency and selectivity)

1. H, OMe, Me, aliphatic amines

2. Piperdine, piperizines, morpholines, thiomorpholines

(H, OMe, piperidines, and piperizines provied optimal potency and selectivity)

Benzenoid ring replaced with pyridine(pyridine analog lost activity)

Page 36 of 124

Table 9. Comparison of the Probe to Project Criteria

No. Property CPDP Requirement Probe

(Avg. IC50)

1 TRPM1 Potency < 5 uM 1.2 uM

2 SKMEL5 Potency < 10 uM 0.4 uM

3 MALME-3M

Potency < 10 uM 0.7 uM

4 A375 Potency > 30 uM >35 uM

5 Primary melanocyte

Potency < 10 uM 7 uM

6 Functional groups

Avoid chemically reactive groups, metabolically labile

groups, pH sensitive or hydrolytically unstable groups

No reactive functionality

7 Solubility Soluble in aqueous buffer 0.4 uM in PBS (pH 7.4, 23°C)

Approximately 160 compound analogs were synthesized during this probe development project,

of which all are reported in PubChem, and only a subset are reported in this report. ML329 is

not part of the current MLSMR HTS library and thus not listed in any PubChem assays. A

number of closely related analogs are present in PubChem with various levels of activity. The

initial hit (MLS000680589/CID 1716436) has been tested in approximately 617 assays and is

active in 48 assays. Six of those assays are part of the MITF project. MLS000680589 was

retested in a number of assays with reported activity of less than 10 uM including: a delayed

death malaria plastid assay (AID 504832), a qHTS assay for inhibitors of fructose-1,6-

bisphosphate aldolase in Giardia lamblia (AID 2451), a Sentrin-specific protease Caspase-6

selectivity assay (AID 488901), an inhibitor in a DnaB-Intein splicing assay (AID 449749),

activator of integrin-mediated alleviation of muscular dystrophy (AID 624291), inhibitor of MBNL-

1-poly(CUG) RNA binding (AID2675), lipid storage modulation in Drosophila S3 cells (AID

2685), inhibition of beta cell apoptosis (AID 449756) and as an activator of Nrf2 (AID 624171).

Many of these are “loss of signal” luciferase assays which could raise concern that there is

modulation of luciferase but activity in some of these assays leads to an increase in luciferase

signal, like the beta cell apoptosis project (AID 449756), rather than decreased signal as seen in

Page 37 of 124

other assays. These results suggest that our lead does not specifically alter luciferase signal but

is working by some other means. To address off-target effects, ML329 was tested against a

panel of 67 targets by Eurofins Panlabs. The only appreciable inhibition observed was against

several human adenosine receptors (Appendix H). The probe compound has potent activity

against several melanoma cell lines (e.g. SK-MEL-5, MALME-3M) and primary melanocytes all

of which require MITF for survival. The Fisher lab has additional melanoma cell lines to test

which will provide further correlative evidence linking compound activity to MITF dependency.

4.1 Comparison to existing art and how the new probe is an improvement

Investigation into relevant prior art entailed searching the following databases: SciFinder and

the Thomson Reuters Integrity. The search terms applied and hit statistics for the prior art

search are provided in Table A2 (Appendix G). Abstracts were obtained for all references

returned and were analyzed for relevance to the current project. For all references that were

deemed relevant, the articles were analyzed and the results are summarized below. The

searches are current as of October 9, 2012.

Page 38 of 124

Figure 7. Reported inhibitors of MITF

Since the start of the project, a modest amount of small-molecule prior art has been reported in

the scientific literature, especially in the year 2012. However, it is not known if these small-

molecule MITF inhibitors recapitulate genetic manipulation or deletion of MITF. Prior to the start

of this probe project, the pilot screen identified a compound, parthenolide, that decreased

TRPM-1 expression but was inadequate in the other cell-based assays because of a lack of

killing in MITF-dependent cell lines. It was used as the positive control for the primary assay for

this project. This compound does not show selectivity to MITF and potently kills some MITF-

independent melanoma and other cancer cell lines. 2-amino-3H-phenoxazin-3-one (APO) was

shown to reduce melanogenesis and the expression of MITF (18). We tested APO and found

that it potentially inhibited TRPM-1 promoter activity (IC50= 2uM) but also killed A375 cells (IC50=

9 uM) suggesting a mode of action nonspecific to MITF. Another report described “compound-

17”, a small molecule that inhibited MITF binding to E-box DNA motifs that are found in the

promoters of several pigment cell-specific genes like TRPM-1 (19). Compound-17 showed

Page 39 of 124

activity with 1 mM of compound in an electrophoretic mobility shift assay (EMSA) but did not

alter TRPM-1 protein levels with up to 50 uM of compound after 72 hours of treatment.

Although we have not measured TRPM-1 protein levels, we have measured promoter activity in

the luciferase assay and mRNA transcript level by qPCR. We detect changes with low

micromolar amounts of ML329 after 24 hours of treatment in both the luciferase and qPCR

TRPM-1 assays (Figure 5A and 8A). One lead compound (CID 9616928) was eliminated

because of lack of potency in SK-MEL-5 cell cytotoxicity, but notably, is similar to the HDAC

inhibitor, panobinostat/LBH589 (CID 6918837), which has been shown to reduce MITF

expression levels and reduce melanoma tumor growth in vivo (20). LBH589 potently reduced

TRPM-1 expression in the luciferase reporter assay but failed to meet the other probe criteria for

the remaining cell-based assays. In particular, LBH589 was not potent enough in SK-MEL-5

cells and led to over 50% reduced viability in A375 cells. A number of compounds have been

reported to inhibit melanogenesis or MITF but none have been shown to directly interact with

MITF and so were not tested against ML329. Several of these substances with low micromolar

activity are listed in Table 10.

Page 40 of 124

Table 10. Compounds reported to inhibit MITF

Compound

Decreased

MITF protein

or mRNA

Mechanism

Concentration

(uM)

Reference

LBH589 protein HDACs 2 20

compound 17 protein E-box binding 50 19

APO protein unknown 1.3 18

hirseins A & B protein MAPK 0.1 21, 22

baicalein protein ERK 1-10 23

propafenone protein cAMP 1-10 24

1-O-MFF protein ERK/Akt 5 25

protocatechuic

acid

protein unknown 20 26

ciglitazone protein CXCL1 10 27

fisetin both Wnt/Catenin 40-80 28

momilactone B Protein PKA <10 uM 29

dihydrofuran 4 protein E-box binding 1-30 30

cordycepin mRNA ERK/Akt 5-20 31

tumerone mRNA CREB 5-40 32

AVS-1357 Protein unknown 10 33

haginin A protein ERK 34

Anemonin mRNA unknown 50 35

taurine Protein ERK 36

PS & VS

dipeptides

Protein ERK 20 37

3,4-DHAP Protein unknown 10 38

terrein Protein ERK 10-100 39

KHG22394 Protein ERK 25 40

cardamonin protein Wnt/Catenin Up to 20 41

glycoside 3 Protein unknown 10 42

4.2 Mechanism of Action Studies

The assays utilized in this project were phenotypic, cell-based assays that leave some

ambiguity to how ML329 works in cells. The assays used in this project only show

circumstantial evidence of ML329 regulating MITF and its molecular pathways. The compound

could be acting on an upstream activator of MITF since we see a reduction of MITF transcription

and several of its target genes by qPCR (Figure 8B, AID 651773). MITF expression is

regulated by a number of transcriptional regulators including: MITF itself, BRAF, WNT signaling,

Page 41 of 124

SOX10, CREB, and Pax3 (2). ML329 reduced the expression of multiple MITF target genes

suggesting a mechanism that impacts a broader profile of MITF targets, including both

melanogenesis and cell cycle genes such as CDK2 (Figure 8C). ML329 consistently lowered

the expression of several MITF target genes including: melastatin (TRPM-1), dopachrome

tautomerase/tyrosinase-related protein-2 (DCT), the cell cycle regulator cyclin-dependent

kinase-2 (CDK2) and melan-A (MLANA/MART1). The primary screen was carried out in a

BRAF(V600E) mutated melanoma cell line, SK-MEL-5. It is quite clear that ML329’s activities

are very different from those of BRAF inhibitors, something studied in the Fisher lab quite

extensively. BRAF-MEK-MAPK lead to proteolysis of MITF, and thus MAPK pathway

suppression leads to MITF stabilization/up-regulation. We have observed (and published) that

multiple MITF target genes are up-regulated following BRAF inhibitor treatments. In contrast we

find that ML329 suppresses both MITF and multiple of its transcriptional target genes. We

believe that MITF up-regulation following BRAF suppression represents a survival mechanism

that limits efficacy of BRAF targeted therapies. Therefore, it is plausible that concurrent use of

MITF antagonists (like ML329) may offer significant benefit in combination with BRAF inhibitors.

Proper determination of mechanism of action will require a number of different studies, some of

which are outlined in Section 4.3.

Page 42 of 124

Figure 8. qPCR for MITF and several target genes

MLS004556029 (CID 12387471, SID 144221520) was tested across a range of concentrations up to 35 uM

in SK-MEL-5 melanoma cells for multiple qPCR assays. Concentration response curves were generated

with Genedata Screener Condeseo and show normalized percent activity for the individual doses based

upon fold change. An increase in fold change correlates with a reduction in gene expression. TRPM-1

qPCR assay (AID 651770), EC50= 0.16 uM (A); MITF qPCR assay (AID 651773), EC50=0.16 uM (B); CDK2

qPCR assay (AID 651772), EC50= 0.5 uM (C) DCT qPCR assay (AID 651771), EC50 = 0.1 uM (D) and

MLANA qPCR assay (AID 651795), EC50= 0.5 uM (E). =replicate 1, Δ=replicate 2

Page 43 of 124

4.3 Planned Future Studies

Figure 9. Suggested medicinal chemistry to improve cellular activity.

Our recommendations for future SAR expansion around the probe (ML329) involve the

following: (1) substitute the anilino nitrogen with various alkyl or acyl groups, (2) append the

sulfonamide nitrogen with mono- and bis-substituted amines (i.e., methyl, morpholine,

piperidine, or pyrrolidine), (3) move the sulfonamide around the ring to which it is currently

attached, (4) add substituents around the ring system adjacent to the sulfonamide, (5) replace

the aniline-benzenoid moiety with other heterocyclic rings, (6) substitute the C3-position of the

naphthoquinone ring system with methyl and methoxy, (7) replace the benzenoid ring of the

naphthoquinone core with various heterocyclic rings (i.e., pyridine and piperidine), and (8)

substitute the benzenoid ring of the naphthoquinone core with mono- and multi- substituted

electron-withdrawing and/or electron-donating groups, as well as groups to probe steric

requirements.

ML329 was tested with three melanoma cell lines, primary melanocytes, the TRPM-1 reporter

cell line and A375 cells. Only one of these cell lines, A375, does not rely upon MITF for

proliferation. There are a number of other melanoma cell lines available (e.g. M14, UACC62,

UACC257, 501mel, SK-MEL-28, & SK-MEL-2) and the probe will be tested in these other

melanoma cell lines. In addition, MITF has been implicated in clear cell sarcoma and ML329

will be tested in tumor-derived sarcoma cell lines to determine if it maintains activity in a

different disease context (4). ML329 was tested with primary melanocytes from a single human

donor and will be tested with other donor-derived melanocytes to verify consistency across

several genetic backgrounds.

Substitute with H, Alk, Acyl

Mono‐ and bis‐substituted amines, esp. H, Me, morpholine, piperidine, etc.

Substituted with H, Me, OMe, and more promising amines

1. Move sulfonamide around the ring2. Add additional substituents3. Heteroaromatic rings with pattern of:

1. Other heterocyclic rings, e.g., pyridine and piperidine

2. Mono‐ and multi‐substituted with EDG or EWG

Page 44 of 124

For mechanism of action studies, ML329 could be tested using biophysical experiments to

determine direct binding of the compound to the MITF protein. Differential scanning fluorimetry

(DSF) or thermal shift is a means of detecting binding of a ligand to purified protein. This

technique is routinely used at the Broad Institute and could be applied to MITF. ML329 leads to

a decrease in the expression of MITF and a number of target genes. It is not known if the DNA

binding capacity of MITF is impaired. ML329 could prevent binding of MITF to E-box domains

within promoter elements. An EMSA experiment could address whether binding of MITF to E-

box DNA sequences are impacted by the presence of the compound. A weak MITF inhibitor

has reported activity in an MITF/E-box EMSA (19). A recurrent mutation in MITF has been

associated with familial and sporadic melanoma. This mutation correlates with impaired

sumoylation (20). We can test the compound in the context of this mutation to determine if the

potency is altered or if inhibition is maintained with this mutant version of MITF.

A gene-expression profiling experiment of SK-MEL-5 cells in the absence or presence of the

ML329 will be performed. We plan to test ML329 in a Luminex-based platform called L1000

performed at the Broad Institute where gene expression of 1000 genes is measured after 6 and

24 hours of compound treatment in a panel of 20 cell lines as part of the LINCS program

(https://commonfund.nih.gov/LINCS/). Tools developed at the Broad Institute, such as

GenePattern and Gene Set Enrichment Analysis (GSEA), will be applied to determine the

comparative markers responsible for the greatest difference between the different states. A

computational method also developed at the Broad called the Connectivity Map, or cmap, that

facilitates target identification through the analysis of small-molecule signatures will also be

applied (43). This tool uses gene-expression profiles in a systematic approach to enable the

discovery of functional connections between diseases, genetic perturbations, and small-

molecule perturbations.

A number of melanoma models that utilize xenografts on rodents are used to determine the

capacity of a drug or probe on melanoma (44). These studies can be done by topical

administration of the test substance onto the tumor or intraperitoneal injection. We can evaluate

ML329 in an analogous tumor graft model.

Page 45 of 124

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Appendix A: Assay Summary Table

Table A1. Summary of Completed Assays and AIDs

PubChem AID No.

Type Target Concentration

Range (µM) Samples Tested

488944 Summary MITF Inhibitor project NA NA

488899 Cell-based TRPM-1 promoter activity assay 12.5 331,578

493177 Cell-based TRPM-1 promoter activity assay 0.015-35 1,241



493240 Cell-based SK-MEL-5 cytotoxicity assay 0.015-35 1,280

540335 Cell-based A375 cytotoxicity assay 0.015-35 1,280

493191 Cell-based MALME-3M cytotoxicity assay 0.015-35 1,280

540348 Cell-based TRPM-1 promoter activity assay 0.015-35 29










540347 Cell-based SK-MEL-5 cytotoxicity assay 0.015-35 30



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540346 Cell-based A375 cytotoxicity assay 0.015-35 1,280

624489 Cell-based A375 cytotoxicity assay 0.00006-35 107








540339 Cell-based MALME-3M cytotoxicity assay 0.015-35 30





651773 Cell-based SK-MEL-5 qPCR for MITF 0.00006-35 35

651770 Cell-based SK-MEL-5 qPCR for TRPM-1 0.00006-35 35

651772 Cell-based SK-MEL-5 qPCR for CDK2 0.00006-35 35

651771 Cell-based SK-MEL-5 qPCR for DCT 0.00006-35 35

Page 52 of 124

651795 Cell-based SK-MEL-5 qPCR for MLANA 0.00006-35 35

651920 Cell-based Primary melanocyte cell viability 0.00006-35 33

NA= not applicable

Page 53 of 124

Appendix B: Detailed Assay Protocols

SK-MEL-5 TRPM-1 Luciferase Reporter Assay (2084-01)

SK-MEL-5/TRPM1Luc Culture Medium:

DMEM (High Glucose, HEPES, Phenol Red), Invitrogen Catalog No. 12430-047

Fetal Bovine Serum (10%), Thermo-Hyclone Catalog No. SH30071.03

Pen-Strep-Glutamine (1%), Invitrogen Catalog No. 10378-016

Hygromycin (250 ug/mL), Invitrogen 10687-010

SK-MEL-5/TRPM1Luc Plating Medium: DMEM (High Glucose, no Phenol Red), Invitrogen

Catalog No. 31053-036, Fetal Bovine Serum (10%), Thermo-Hyclone Catalog No. SH30071.03,


Steady Glo Promega Catalog No. E2550

Parthenolide Enzo Catalog No. BML-T113-0250

SK-MEL-5/TRPM1Luc cells were maintained in DMEM (10%FBS, 1% Pen-Strep-Glutamine,

250 ug/mL Hygromycin). Cells were fluid changed every 3 days and/or split upon reaching

100% confluency. For the primary HTS, cells were thawed at 4 million cells per Falcon T175

flask. After 3 days, the cells were fluid changed. After 3 more days, the cells were passed to a

Corning triple flask (10-15 million cells) and plated after 3 days in the triple flask.

Day 1

1. Plate TRPM-1 luc/SKMEL5 cells at 2000 per well in 30 uL media (phenol red free DMEM/10% Fetal Bovine Serum/Penicillin/Streptomycin/L-Glutamine)

2. Use Corning white 384-well, square, opaque-bottomed plates (Corning Catalog No. 8867BC)

Day 2

3. Pin 100 nL compound/DMSO solution (Cybi Well) into assay plates. (For HTS, required sentinel pinning with the positive control, parthenolide (6 mM))

4. Incubate 24 hours at 37˚C in Liconic incubator. Day 3

5. Add 20 uL 100% Promega SteadyGlo per well with Thermo Combi fluid transfer apparatus.

6. Shake 15 seconds on “big bear” plate shaker and incubate at room temperature for 5 minutes.

7. Read on the Perkin-Elmer EnVision plate reader with ultra-sensitive luminescence (US LUM) settings for 0.5 sec per well

SK-MEL-5 Cytotoxicity Assay (2084-02)

Page 54 of 124

SK-MEL-5 Cells ATCC Catalog No. HTB-70, lot 58483232, passage 28

SK-MEL-5 Culture Medium:




SK-MEL-5 Plating Medium:

DMEM (High Glucose, no Phenol Red), Invitrogen Catalog No. 31053-036



SK-MEL-5 cells were maintained in DMEM (10%FBS, 1% Pen-Strep-Glutamine). Cells were

fluid changed every 3 days and/or split upon reaching 100% confluency. For secondary assays,

cells were thawed at 4 million cells per Falcon T175 flask. After 3 days, the cells were fluid

changed, after 3 more days cells were passed to a Corning Triple flask (10-15 million cells) and

plated after 3 days in the triple flask.

Day 1

1. Plate SK-MEL-5 cells at 3,000 per well in 30 uL media (phenol red free DMEM/10% Fetal Bovine Serum/Penicillin/Streptomycin/L-Glutamine)


Day 2



5. Add 20 uL 100% Cell Titer GLO per well with Thermo Combi fluid transfer apparatus. 6. Shake 15 seconds on “big bear” plate shaker and incubate at room temperature for 5

minutes. 7. Read on the Perkin-Elmer EnVision plate reader with luminescence (LUM) settings for

0.1 sec per well.

A-375 Cytotoxicity Assay (2084-03)

A-375 Cells ATCC Catalog No. CRL-1619, Lot No. 58463364, passage 166

A-375 Culture Medium:



Page 55 of 124


A-375 Plating Medium:

DMEM (High Glucose, no Phenol Red), Invitrogen Catalog No. 31053-036



A-375 cells were maintained in DMEM (10%FBS, 1% Pen-Strep-Glutamine). Cells were fluid

changed every 3 days and/or split upon reaching 90% confluency. For secondary assays, cells

were thawed at 2 million cells per Falcon T175 flask. After 3 days, cells were passed to a

Corning Triple flask (6-8 million cells) and plated after 3 days in the triple flask.

Day 1

1. Plate A-375 cells at 3,000 per well in 30 uL media (phenol red free DMEM/10% Fetal Bovine Serum/Penicillin/Streptomycin/L-Glutamine)


Day 2



5. Add 20 uL 100% Promega Cell Titer GLO per well with Thermo Combi fluid transfer apparatus.


7. Read on the Perkin-Elmer EnVision plate reader with luminescence (LUM) settings for 0.1 sec per well.

MALME-3M Cytotoxicity Assay (2084-04)

MALME-3M Cells ATCC Catalog No. HTB-64, Lot No. 58483222, passage 26

MALME-3M Culture Medium:

IMDM (High Glucose, Phenol Red), ATCC Catalog No. 30-2005



MALME-3M Plating Medium:

IMDM (no Phenol Red), Gibco Catalog No. 21056-02


Page 56 of 124


MALME-3M cells were maintained in IMDM (20%FBS, 1% Pen-Strep-Glutamine). Cells were


cells were thawed at 6 million cells per Falcon T175 flask. After 3 days, cells were fluid

changed, after 3 more days cells were passed to a Corning Triple flask (15-18 million cells) and


Day 1

1. Plate MALME-3M cells at 3,000 per well in 30 uL media (phenol red free IMDM/10% Fetal Bovine Serum/Penicillin/Streptomycin/L-Glutamine)


Day 2

3. Pin 100 nL compound/DMSO solution (Cybi Well) into assay plates, including the positive control.





Cell proliferation assay with primary human melanocytes (2084-06)

Primary human neonatal melanocytes were isolated from discarded foreskins by gentle dispase

treatment and grown in TIVA media (Ham’s F10 media supplemented with 7% FBS,

penicillin/streptomycin/glutamine, 0.1mM IBMX, 50ng/mL TPA, 1µM Na3VO4 and 1µM dbcAMP).

Cells were passaged using Accutase (Sigma Catalog #A6964-100ML) for gentle treatment and

generation of a single cell suspension.

Day 1

1. Plate primary melanocytes at 3,000 per well in 30 uL media (TIVA media) 2. Use Corning white 384-well, square, opaque-bottomed plates (Corning Catalog No.

8867BC) Day 2

3. Pin 100 nL compound/DMSO solution (Cybi Well) into assay plates. (pinning with the positive control, parthenolide (18 uM final concentration))


Page 57 of 124




qPCR Assay for Target Gene Expression (MITF: 2084-05, TRPM1: 2084-09, CDK2: 2084-

11, DCT: 2084-12, MLANA: 2084-13)

SK-MEL-5 Cells ATCC Catalog No. HTB-70, lot 58483232, passage 28

SK-MEL-5 Culture/Plating Medium:




Parthenolide Enzo Catalog No. BML-T113-0250

Cells to CT Bulk Lysis Solution Ambion Catalog No. 4391851C

Cells to CT Bulk RT Reagents Ambion Catalog No. 4391852C

Light Cycler 480 Probes Master Roche Catalog No. 4887301001

Human GAPD (GAPDH) Endogenous Control VIC/MGB probe/primer limited Applied

Biosystems Catalog No. 4326317E

Target Gene FAM probe/primer sets Human MITF FAM probe/primer Applied Biosystems Catalog No. 4331182 Hs01117294_m1 Human TRPM1 probe/primer Applied Biosystems Catalog No. 4331182 Hs00170127_m1 Human CDK2 probe/primer Applied Biosytems Catalog No. 4331182 Hs01548894_m1 Human DCT probe/primer Applied Biosystems Catalog No. 4331182 Hs01098278_m1 Human MLANA probe/primer Applied Biosystems Catalog No. 4331182 Hs00194133_m1

SK-MEL-5 cells were maintained in DMEM (10%FBS, 1% Pen-Strep-Glutamine). Cells were


cells were thawed at 4 million cells per Falcon T175 flask. After 3 days, the cells were fluid

changed, after 3 more days cells were passed to a Corning triple flask (10-15 million cells) and


Page 58 of 124

Day 1

1. Plate SK-MEL-5 cells at 4,000 per well in 30 uL media (DMEM/10% Fetal Bovine Serum/Penicillin/Streptomycin/L-Glutamine)


Day 2

3. Pin 100 nL compound/DMSO solution (Cybi Well) into assay plates. (in plate positive control, parthenolide (6 mM))

4. Incubate 24 hours at 37˚C in Liconic incubator Day 3

Cell Lysis

5. The medium is aspirated from assay plates and the cells are washed twice (100 uL PBS) using the ELX405 Plate Washer (Biotek).

6. The assay plates are flipped upside down and centrifuged at 1000 rpm for 2 minutes to remove the excess liquid.

7. 10 uL of Lysis solution with DNase I (Ambion, from Cell to CT Lysis Mix) is added to each well using the MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific).

8. Each assay plate is then shaken for 2 minutes and incubated for an additional 8 minutes at room temperature.

9. 1 uL of stop solution (Ambion, from Cell to CT Lysis Mix) is added with the Multidrop Combi-nL (Thermo Scientific) and the assay plate is centrifuged at 1000 rpm for 2 minutes.

Reverse Transcription (RT) Mix

Table 1.

Component Amount per reaction

2X RT Buffer 5 uL

20X RT Enzyme Mix 0.5 uL

Nuclease-Free Water 2.5 uL

10. 8 uL of RT mix is dispensed into each well of a RT assay plate (Axygen, PCR-384 RGD

C). 11. 2 uL of the lysed cells are transferred into RT assay plate using Vario transfer unit (Cybi

Well). 12. The RT assay plates are incubated at 37˚C for 1 hour and the reverse transcriptase is

inactivated by incubating the plates for 1 minute at 95˚C. 13. cDNA is stored at -80˚C until ready for qPCR analysis

Day 4

Page 59 of 124

qPCR Master Mix

Table 2.

Component Amount per reaction

2X Roche Master Mix 2.5 uL

20X FAM Target Gene Taqman probe/primer 0.125 uL

20X VIC GAPDH Taqman probe/primer 0.125 uL

PCR water 1.25 uL

14. 4 uL/well of qPCR master mix is dispensed in PCR plate (Roche Light Cycler 480 Multiwell Plate 384, Catalog No. 04 729 749 001) using the Multidrop Combi-nL (Thermo Scientific).

15. 1 uL/well of RT DNA is transferred in the 4 uL/well PCR plate. 16. The PCR plates are centrifuged for 2 minutes at 1000 rpm. 17. PCR is performed using Thermo Cycler (Roche Light Cycler 480 II) with Macro Protocol:

Step Temperature Time

1. 95˚C 10 minutes

2. 95˚C 10 seconds

3. 60˚C 30 seconds

Step 2 and 3 (55 cycles)

4. 40˚C 30 seconds

Data Analysis

For the primary screen and other assays, negative-control (NC) wells and positive-control (PC)

wells were included on every plate. The raw signals of the plate wells were normalized using the

'Stimulators Minus Neutral Controls' or the 'Neutral Controls' method (when no positive control

was available) in GeneData Screener Assay Analyzer (v7.0.3 & v10.0.2). The median raw signal

of the intra-plate NC wells was set to a normalized activity value of 0, while the median raw

signal of the intra-plate PC wells was set to a normalized activity value of 100. Experimental

wells were scaled to this range, resulting in an activity score representing the percent change in

signal relative to the intra-plate controls. The mean of the replicate percent activities were

presented as the final ’PubChem Activity Score’. The ’PubChem Activity Outcome’ class was

assigned as described below, based on an activity threshold of 70%:

Activity_Outcome = 1 (inactive), less than half of the replicates fell outside the threshold.

Activity_Outcome = 2 (active), all of the replicates fell outside the threshold, OR at least

half of the replicates fell outside the threshold AND the ’PubChem Activity Score’ fell

outside the threshold.

Activity_Outcome = 3 (inconclusive), at least half of the replicates fell outside the

threshold AND the ‘PubChem Activity Score did not fall outside the threshold.

Page 60 of 124

Appendix C: Experimental Procedures for the Synthesis of the Probe

General synthesis and analysis experimental details: All reagents were used as received from

commercial suppliers. The 1H NMR spectra were recorded on a 400 MHz Bruker Avance

spectrometer equipped with a broadband observe probe or a 500 MHz Bruker AVIII

spectrometer equipped with a dual cryoprobe. The 13C NMR spectra were recorded on a 500

MHz Bruker AVIII spectrometer equipped with a dual cryoprobe (at 125 MHz). Column

chromatography separations were performed using the Teledyne Isco CombiFlash Rf using

RediSep Rf silica gel or RediSep Rf C18 High Performance Gold columns. The analytical RPLC

method used an Agilent 1200 RRLC system with UV detection (Agilent 1200 DAD SL) and mass

detection (Agilent 6224 TOF). The analytical method conditions included a Waters Aquity BEH

C18 column (2.1 × 50 mm, 1.7 µm) and elution with a linear gradient of 5% acetonitrile in pH 9.8

buffered aqueous ammonium formate to 100% acetonitrile at 0.4 mL/min flow rate. Compound

purity was measured on the basis of peak integration (area under the curve) from UV-vis

absorbance at 214 nm, and compound identity was determined on the basis of mass spectral

and NMR analyses. All compounds used for biological studies have purity of ≥92%.

ML329 CID 12387471 SID 144221520

4-((1,4-Dioxo-1,4-dihydronaphthalen-2-yl)amino)benzenesulfonamide (ML329): Cerium chloride

heptahydrate (36 mg; 97 µmol), followed by 4-aminobenzenesulfonamide (689 mg; 4.00 mmol),

was added to a suspension of naphthalene-1,4-dione (316 mg; 2.00 mmol) in 95% ethanol (8.0

mL). The reaction vial was capped then put into a 75 °C block. After three days the reaction was

cooled to room temperature, then 80 mL of 1.0 M citric acid was added with vigorous stirring.

The insoluble material was collected by filtration, washed with water, and dried in vacuo at 45

°C affording crude product as a rust colored solid (370 mg). A portion of this (225 mg) was

purified by pRPLC yielding 4-((1,4-dioxo-1,4-dihydronaphthalen-2-

yl)amino)benzenesulfonamide as a rust-colored solid (101 mg).

1H NMR (400 MHz, DMSO-d6) δ 9.45 (s, 1H), 8.09 (dd, J = 1.0, 7.6 Hz, 1H), 7.98 (dd, J = 1.1,

7.6 Hz, 1H), 7.92 – 7.79 (m, 4H), 7.60 (m, 2H), 7.37 (s, 2H), 6.33 (s, 1H).

13C NMR (125 MHz, DMSO-d6) δ 183.0, 181.3, 145.2, 141.5, 139.6, 134.9, 132.9, 132.3, 130.4,

127.0, 126.2, 125.3, 122.7, 103.8.

LC-MS (ESI+): Purity at 214 nm is 100%. HRMS: 329.0591 (calcd for C16H13N2O4S = [M+H]+);

329.0594 (found / [M+H]+).

Page 61 of 124

Appendix D: Experimental Procedure for Analytical Assays

Solubility. Solubility was determined in phosphate buffered saline (PBS) pH 7.4 with 1% DMSO. Each compound was prepared in duplicate at 100 uM in both 100% DMSO and PBS with 1% DMSO. Compounds were allowed to equilibrate at room temperature with a 250 rpm orbital shake for 24 hours. After equilibration, samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer. The DMSO samples were used to create a two-point calibration curve to which the response in PBS was fit.

PBS Stability. Stability was determined in the presence of PBS pH 7.4 with 0.1% DMSO. Each compound was prepared in duplicate on six separate plates and allowed to equilibrate at room temperature with a 250-rpm orbital shake for 48 hours. One plate was removed at each time point (0, 2, 4, 8, 24, and 48 hours). An aliquot was removed from each well and analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer. Additionally, to the remaining material at each time point, acetonitrile was added to force dissolution of compound (to test for recovery of compound). An aliquot of this was also analyzed by UPLC-MS.

GSH Stability. Stability was determined in the presence of PBS pH 7.4, 10 uM compound and 50 uM glutathione with 0.1% DMSO. Each compound was prepared in duplicate on six separate plates and allowed to equilibrate at room temperature with a 250-rpm orbital shake for 48 hours. One plate was removed at each time point (0, 2, 4, 8, 24, and 48 hours). An aliquot was removed from each well and analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer. Additionally, to the remaining material at each time point, acetonitrile was added to force dissolution of compound (to test for recovery of compound). An aliquot of this was also analyzed by UPLC-MS.

DTT Stability. Compound was dissolved at 10 µM in PBS/acetonitrile (1/1) at pH 7.4 (1% DMSO) and incubated at room temperature with either no thiol source as a negative control or 50 µM dithiothreitol (DTT). The mixtures were sampled every hour for eight hours or every 8 hours for 48 hours and analyzed by RP HPLC/UV/HRMS. The analytical RP HPLCUV/HRMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 x 50mm, 1.8um) and elution with a linear gradient of 99% water to 100% CH3CN at 0.6 mL/min flow rate. Peaks on chromatograms were integrated using the Waters OpenLynx software. Absolute areas under the curve (214 nm) were compared at each time point to determine relative percent compound remaining in supernatant. The masses of potential adducts were searched for in the samples to determine if any detectable adduct formed. All samples were prepared in duplicate. Ethacrynic acid, a known Michael acceptor, was used as a positive control and was tested in PBS/acetonitrile (1/1).

Plasma Protein Binding. Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology, Rockford, IL) for both human and mouse plasma. Each compound was prepared in duplicate at 5 uM in plasma

Page 62 of 124

(0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 uL) with PBS pH 7.4 added to the other side (350 uL). Compounds were incubated at 37 ºC for 5 hours with a 250-rpm orbital shake. After incubation, samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Plasma Stability. Plasma stability was determined at 37ºC at 5 hours in both human and mouse plasma. Each compound was prepared in duplicate at 5 uM in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). Compounds were incubated at 37ºC for 5 hours with a 250-rpm orbital shake with time points taken at 0 hours and 5 hours. Samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Page 63 of 124

Appendix E: Chemical Characterization Data for Probe

1H NMR Spectrum (400 MHz, DMSO) of the Probe ML329

13C NMR Spectrum (125 MHz, DMSO) of the Probe ML329

Page 64 of 124

UPLC Chromatogram of the Probe ML329

HRMS Chromatogram of the Probe ML329

Page 65 of 124

Appendix F: Chemical Characterization Data for All Analogs

1H NMR Spectrum (400 MHz, CDCl3) of Analog CID1716436

UPLC-MS Chromatogram of Analog CID1716436

Page 66 of 124

1H NMR Spectrum (500 MHz, DMSO) of Analog CID56951838


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Page 119 of 124

Appendix G: Prior Art Search

Table A2. Search Strings and Databases Employed in the Prior Art Search

Search String Database Hits Found

“MITF inhibitor” SciFinder 1 as entered

“MITF inhibitor”

SciFinder 438 containing

concept

“small molecule inhibitors MITF” SciFinder 6

“MITF inhibitor” Thomson Reuters

Integrity 0

“microphthalmia” Thomson Reuters

Integrity 0

“MITF inhibitors” and “2010-“ SciFinder 131

“MITF inhibitors” and “-2010“ SciFinder 199

Page 120 of 124

Appendix H: Eurofins Panlabs LeadProfiling Screen Report for ML329

The following text was provided along with the study results in the report for the

LeadProfilingScreen for ML329.

Study Objective:

To evaluate, in radioligand binding assays, the activity of probe compound ML329

(KUC111774N-02) across a panel of 67 receptors.

Methods:

Methods employed in this study have been adapted from the scientific literature to maximize

reliability and reproducibility. Reference standards were run as an integral part of each assay to

ensure the validity of the results obtained.

Where presented, IC50 values were determined by a non-linear, least squares regression

analysis using MathIQTM (ID Business Solutions Ltd., UK). Where inhibition constant (Ki) are

presented, the Ki values were calculated using the equation of Cheng and Prusoff (Cheng. Y.,

Prusoff, W.H., Biochem. Pharmacol. 22:3099-3108, 1973) using the observed IC50 of the tested

compound, the concentration of radioligand employed in the assay, and the historical values of

the KD of the ligand (obtained experimentally at Eurofins Panlabs, Inc.). Where presented,

the Hill coefficient (nH), defining the slope of the competitive binding curve, was calculated using

MathIQTM. Hill coefficients significantly different than 1.0, may suggest that the binding

displacement does not follow the laws of mass action with a single binding site. Where IC50, Ki,

and/or nH data are presented without Standard Error of the Mean (SEM), data are insufficient to

be quantitative, and the values presented (Ki, IC50, nH) should be interpreted with caution.

Page 121 of 124

Cat # Assay Name Batch* Spec. Rep. Conc. % Inh.

200510 Adenosine A1 324045 human 2 10 µM 46

200610 Adenosine A2A 324044 human 2 10 µM 50

200720 Adenosine A3 324012 human 2 10 µM 15

203100 Adrenergic α1A 324046 rat 2 10 µM 28

203200 Adrenergic α1B 324024 rat 2 10 µM 16

203400 Adrenergic α1D 324047 human 2 10 µM 21

203620 Adrenergic α2A 324048 human 2 10 µM 33

204010 Adrenergic β1 324027 human 2 10 µM 20

204110 Adrenergic β2 324042 human 2 10 µM 10

285010 Androgen (Testosterone) AR 324064 rat 2 10 µM 14

212510 Bradykinin B1 324141 human 2 10 µM 29

212620 Bradykinin B2 324310 human 2 10 µM 11

214510 Calcium Channel L-Type, Benzothiazepine

324113 rat 2 10 µM 20

214600 Calcium Channel L-Type, Dihydropyridine

324317 rat 2 10 µM 30

216000 Calcium Channel N-Type 323997 rat 2 10 µM -3

217030 Cannabinoid CB1 324041 human 2 10 µM 31

219500 Dopamine D1 324037 human 2 10 µM 21

219700 Dopamine D2S 324038 human 2 10 µM 0

219800 Dopamine D3 324039 human 2 10 µM 9

219900 Dopamine D4.2 324230 human 2 10 µM 4

224010 Endothelin ETA 324347 human 2 10 µM 17

224110 Endothelin ETB 324349 human 2 10 µM 17

225510 Epidermal Growth Factor (EGF) 324350 human 2 10 µM 11

226010 Estrogen ERα 324351 human 2 10 µM 5

226600 GABAA, Flunitrazepam, Central 324035 rat 2 10 µM 20

226500 GABAA, Muscimol, Central 324034 rat 2 10 µM 4

228610 GABAB1A 324354 human 2 10 µM 11

232030 Glucocorticoid 324040 human 2 10 µM 5

232700 Glutamate, Kainate 324324 rat 2 10 µM 43

232810 Glutamate, NMDA, Agonism 324326 rat 2 10 µM 29

232910 Glutamate, NMDA, Glycine 324329 rat 2 10 µM 9

233000 Glutamate, NMDA, Phencyclidine 324036 rat 2 10 µM 0

239610 Histamine H1 324052 human 2 10 µM -5



241000 Imidazoline I2, Central 324053 rat 2 10 µM -5

243520 Interleukin IL-1 324013 mouse 2 10 µM 6

250460 Leukotriene, Cysteinyl CysLT1 324130 human 2 10 µM 5

251600 Melatonin MT1 324356 human 2 10 µM 9

252610 Muscarinic M1 324054 human 2 10 µM 7



257010 Neuropeptide Y Y1 324363 human 2 10 µM -6

257110 Neuropeptide Y Y2 324365 human 2 10 µM 5

258590 Nicotinic Acetylcholine 324018 human 2 10 µM 9

Page 122 of 124

258700 Nicotinic Acetylcholine α1, Bungarotoxin

324019 human 2 10 µM -2

260130 Opiate δ1 (OP1, DOP) 324366 human 2 10 µM -6

260210 Opiate κ(OP2, KOP) 324124 human 2 10 µM 19

260410 Opiate μ(OP3, MOP) 324058 human 2 10 µM 1

264500 Phorbol Ester 324067 mouse 2 10 µM 18

265010 Platelet Activating Factor (PAF) 324114 human 2 10 µM 3

265600 Potassium Channel [KATP] 324068 hamster 2 10 µM 10

265900 Potassium Channel hERG 324069 human 2 10 µM -4

268420 Prostanoid EP4 324070 human 2 10 µM 11

268700 Purinergic P2X 324103 rabbit 2 10 µM -2

268810 Purinergic P2Y 324128 rat 2 10 µM -7

270000 Rolipram 324059 rat 2 10 µM 15

271110 Serotonin (5-Hydroxytryptamine) 5-HT1A

324121 human 2 10 µM 21

271700 Serotonin (5-Hydroxytryptamine) 5-HT2B

324061 human 2 10 µM 37

271910 Serotonin (5-Hydroxytryptamine) 5-HT3

324005 human 2 10 µM 5

278110 Sigma σ1 324062 human 2 10 µM 15

279510 Sodium Channel, Site 2 324063 rat 2 10 µM 6

255520 Tachykinin NK1 324358 human 2 10 µM 37

285900 Thyroid Hormone 324137 rat 2 10 µM -4

220320 Transporter, Dopamine (DAT) 324015 human 2 10 µM 2

226400 Transporter, GABA 324353 rat 2 10 µM 9

204410 Transporter, Norepinephrine (NET) 324014 human 2 10 µM 8

274030 Transporter, Serotonin (5-Hydroxytryptamine) (SERT)

324370 human 2 10 µM -5

* Batch: Represents compounds tested concurrently in the same assay(s).

Page 123 of 124

Appendix I: Primary melanocyte viability assay data

Figure A.1

MLS004556029 (CID 12387471, SID 144221520) was tested across a range of concentrations up to 35 uM in a

primary human melanocyte assay. Compounds were incubated for 24 hours and viability measured with CellTiter-

Glo (AID 651920). Concentration response curves were generated with Genedata Screener Condeseo and show

normalized percent activity for the individual doses. IC50 = 7.14 uM. =replicate 1, Δ=replicate 2, ☐=replicate 3

Page 124 of 124

Appendix J: Compounds Provided to Evotec

Table A3. Probe and Analog Information

CID / SID

Broad ID / KU ID

P/A MLSID ML

12387471 / 144221520

BRD-K73037408-001-01-5 /KUC111774N-03

P MLS004556029 ML329

81124 / 144221523

BRD-K29842115-001-07-4 /KUC111337N-02

A MLS004556032 NA

72909 / 144221522

BRD-K75502546-001-01-9 /KUC111359N-02

A MLS004556031 NA

279009 / 144221524

BRD-K48101260-001-01-8 /KUC111761N-02

A MLS004556033 NA

56951846 / 144221521

BRD-K93744531-001-01-2 /KUC111358N-02

A MLS004556030 NA

56928031 / 135611193

BRD-K16604218-001-01-2 /KUC111109N

A MLS004556034 NA

A = analog; NA= not applicable; P = probe

Documents

A Small Molecule Inhibitor of the MITF Molecular Pathway