Vaistų kūrimas Lietuvoje ir pasaulyje

Daumantas Matulis

BVTS vedėjas, Vilniaus universiteto Biotechnologijos Institutas

Introduction (Miguel Fernandez)

• Drug

– Safe

– Effective

– Stable

– Soluble

– Synthesizable

– Novel

THE DISCOVERY OF A NEW DRUG

3

~ $1-2 billion

Preclinical Development

Lead Optimization

Lead Discovery

Target Discovery

Clinical Trials

DRUG LAUNCH

~12 – 15 years

19 FDA approved drug products containing new chemical entities in 2012

THE MARKET

4 http://www.forbes.com/sites/matthewherper/2012/02/10/the-truly-staggering-cost-of-inventing-new-drugs/

Therapeutic targets •~30000 genes in human genome

•Therapeutic targets?

Therapeutic targets

Hopkins, AL; Groom, CR (2002) Nat. Rev. Drug Discov. 1: 727

Therapeutic targets

Hopkins, AL; Groom, CR (2002) Nat. Rev. Drug Discov. 1: 727

Neuraminidase (NA) Influenza virus H1N1 neuraminidase

helps viruses to be released from a host cell. Influenza virus membranes contain two glycoproteins: hemagglutinin and neuraminidase. While the hemagglutinin on the surface of the virion is needed for infection, its presence inhibits release of the particle after budding. Viral neuraminidase cleaves terminal neuraminic acid (also called sialic acid) residues from glycan structures on the surface of the infected cell. This promotes the release of progeny viruses and the spread of the virus from the host cell to uninfected surrounding cells.

Glu276 Arg276 Arg292

Glu119 Glu227

Rational design Neuraminidase

Sialic acid

Ki = 1.0 mM

Glu119 Glu227

Ki = 0.1 nM

zanamivir

Zanamivir

11

12

ThermoFluor technology

30000

50000

70000

90000

110000

130000

150000

170000

190000

40 50 60 70 80 90

Temperature, °C

Flu

ores

cen

ce I

nte

nsi

ty, C

ou

nts

Without

Ligand

With

Ligand

Tm

13

ThermoFluor® plate

14

Observed Tm↑ and Tm↓

• Red – stabilizers

• Blue – destabilizers

15

Ample thermodynamic data

DRUG

DESIGN

Biothermo-dynanics

Molecular biology

Organic synthesis

Molecular modeling

O

S

SO

ONH

2

NH

N

O

O

17

O

O

NH2

O

O

O

NH

O

OOH

NH

OH

OH

O

O

ClO

O

N

N S

O

Cl

OH

OH

N

N S

O

OH

OH

N

N S

O

Cl

OH

OH

ON

N S

O

Cl

OH

OH

OH

OH

O

O

O

OH

OH

O

O

O

O

N

N

O

NH

O

OH

OH

N

N N

N

Cl

NH2 N

O

17-AAGRadicicol

ICPD47

ICPD62

ICPD34ICPD26

RICH243 (ent-40b) RICH239 (40b)

NVP-AUY922

BIIB021

5515 SacI (2)HisTag 5329...5346

LacO 5249...5271T7 5230...5249

6241 SacI (2)

Hsp90wt 5317...7575

pL0029 pET15b-

Hsp90wt8072 bp

AmpR 407...1066

ColE1 origin 1164...1846

0.0 0.5 1.0 1.5 2.0

-16

-12

-8

-4

0

-0.15

-0.10

-0.05

0.00

0 20 40 60 80

Time, min

mca

l/se

c

Molar Ratio

kca

l/m

ole

of

inje

cta

nt

THE DEPARTMENT PROFILE

Department of Biothermodynamics and Drug Design

• Head of department dr. Daumantas Matulis

• The Team of Molecular and Cellular Biology

• The Team of Organic Synthesis

• The Team of Biophysics

• The Team of Molecular Modelling

13 scientists (Ph.D.) and 11 students

18

COMPETITION

19

Our strengths: Know-how Largest collection of calorimeters in Eastern Europe

Competitors/supporters: Janssen Research & Development , LLC, USA Other laboratories working with calorimetry

20/30

ScanBalt Forum and Biomaterials Days September 23- 26, 2008, Vilnius

Thermal Analysis and Biothermodynamics Conference in Vilnius, Lithuania

CEEC-TAC2 27-30 August 2013 Vilnius, Lithuania

www.ceec-tac.org

21

Hsp90 as an anticancer target

22

Hsp90 structure

23

Sti1

Aha1

ND

CR

MD

CD

Lid ATP

Open Closed

p23

Hessling et al. Nature Struct. Biol. 2009 Mickler et al. Nature Struct. Biol. 2009

Ali et al. Nature 2006

Hsp90 mechanism

24/40

Hsp90 inhibitors in clinical trials

Hsp90 inhibition

Prostate cancer

IPI-504 Ph.II

Udai Banerji, 2009; Marco A. Biamonte et al. 2010; Peyrat JF, Messaoudi S, Brion JD, Alami M 2010 and oth.

Colon cancer

Ovarian and endometrial

cancer

KOS-953 Ph.II

Chronic myeloid leukemia

BIIB021 Ph.I KW-2478 Ph.I STA9090 Ph.II

Multiple myeloma

KW-2478 Ph.I ►II KOS-953 Ph.II ►III with Bortezomib Ph.II

Other hematologic malignancies

IPI-493 Ph.I NVP-AUY922 Ph.II with Bortezomib Ph.II

STA9090 Ph.II

Lung cancer

IPI-504 Ph.II STA9090 Ph.II

Rheumatoid Arthritis

SNX-4414

Parkinson’s disease

Alzheimer’s disease

Fungal infections

Sarcoma

IPI-504 Ph.I ►II

Chronic lymphocytic leukemia

CNF1010 Ph.I BIIB021 Ph.I

KW-2478 Ph.I

Metastatic myeloma

IPI-504 Ph.II

Solid tumors

KOS-1022 Ph.I BIIB021 Ph.I

NVP-AUY922 Ph.II SNX-5422 Ph.I STA9090 Ph.II

MPC-3100 Ph.I AT-13387 Ph.I

Renal cancer

KOS-953 Ph.II

Melanoma

Gastrointestinal stromal tumors

IPI-504 Ph.III BIIB021 Ph.II

with Aromasin Ph.II

NVP-AUY922 Ph.II STA9090 Ph.II

Squamous cell cancers

Breast cancer

IPI-504 Ph.II with Trastuzumab Ph.II

KOS-953 Ph.II KOS-1022 Ph.II

with Trastuzumab, Paclitaxel Ph.II

BIB021 Ph.I with Aromasin Ph.II

NVP-AUY922 Ph.II

Malaria

25

ICPD 26 ICPD 34 ICPD 39

ICPD 72 / VER-49009

ICPD 47 ICPD 62

Hsp90 inhibitors of ICPD series

ICPD 60

NN

S

ClOH

OH

O

NN

S

ClOH

OHN

NS

ClOH

OH

O

O

NN

S

ClOH

OH

O

O

NH

CH3

NNH

OH

OH

OCl

NN

S

OH

OH

O

Cl

NN

S

OH

OH

O

26/84

Intrinsic binding thermodynamics of ICPD inhibitors

27

TF for high precision of Kb

28

Linked protonation effects

29

Intrinsic energetics (SAR) of ICPD inhibitors

30

Hsp90 slopiklių tyrimai su vėžinėmis ląstelėmis

MTT metodas

1. Auginamos ląstelės su skirtingomis junginio koncentracijomis.

2. Baigus auginti, įpilama MTT dažo, kurį gyvybingos ląstelės metabolizuoja į mėlyną produktą. Jis tirpinamas DMSO.

3. Matuojama absorbcija, skaičiuojama IC50.

Galvos ir kaklo vėžys Gaubtinės žarnos vėžys Plaučių vėžys

Galvos ir kaklo vėžys Gaubtinės žarnos vėžys Krūties vėžys

Kiaušidžių vėžys Plaučių vėžys Imortalizuotos krūties audinio ląstelės

2011 m. ICPD47 tyrimas VU Onkologijos institute

allograft metodika (pelėms įskiepytos pelių vėžio MH22a ląstelės )

hibridinės C57BL/6xDBA/2 mot. lyties pelės, 19-22 g

Hsp90 slopiklių priešvėžinio veiksmingumo tyrimai

L

B

H

2012 m. ICPD34, ICPD47 ir NVP-AUY922 tyrimai VU Onkologijos

institute

allograft metodika (pelėms įskiepytos pelių vėžio MH22a ląstelės )

hibridinės C57BL/6xDBA/2 mot. lyties pelės, 19-22 g

2012 m. ICPD34, ICPD47 ir ICPD62 tyrimai IDIBELL, Barselonoje

xenograft metodika (pelėms įskiepytos žmogaus gaubtinės žarnos vėžio HCT116 ląstelės )

nudinės vyr. lyties pelės, 27-34 g

Pelių išgyvenamumas

Pelių išgyvenamumas

Carbonic anhydrase (CA)

38

39/84

Carbonic anhydrases (CA) – Zn containing enzymes

catalyze reaction:

CO2 + H2O ↔ HCO3- + H+

Humans have 12 active CA isoforms involved in numerous phiosiological and pathological processes:

Cancer, glaucoma, obesity, neural…

CAs in cancer

Nordfors et al. BMC Cancer 2010, 10:148

Medulloblastomas - highly aggressive pediatric brain tumors A - no immunoreaction for CA IX. the tumour in B is strongly positive. C - CA XII-positive immunoreactivity in tumour cells. D - CA II-positive immunostaining is confined to the endothelium of small blood vessels (arrows).

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Reduction in tumor size

Chiche, J . et al. Cancer Res, 2009, 69, 358-68

Inducible invalidation of CAIX and CAXII reduces the rate of xenograft tumor growth.

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All CAs have been purified in the lab

Purified in large quantities,

other CAs in small quantities

1 2 3 4 5 6 7 8 1. MW. 2. CA1. 3.CA2. 4. CA7(pL0093) 5.CA12. 6. CA13. 7. CA4. 8. CA7(pL0137). 10mg/lane

42

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 1000 2000 3000 4000 5000

t, s

µca

l/s

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 1000 2000 3000 4000 5000

t, s

µca

l/s

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 1000 2000 3000 4000 5000

t, s

µca

l/s

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 1000 2000 3000 4000 5000

t, s

µca

l/s

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 1000 2000 3000 4000 5000

t, s

µca

l/s

-0.4

-0.35

-0.3

-0.25

-0.2

-0.15

-0.1

-0.05

0

0.05

0 1000 2000 3000 4000 5000

t, sµ

ca

l/s

CAXIII-TFMSA, tris buffer CAXIII-TFMSA, phos buffer

pH 6.0

pH 7.5

pH 9.0

43

CA XIII and CA XII w EZA ITC

5 6 7 8 9 10

-70

-60

-50

-40

-30

-20

(c)

TrisbH

ob

s (

kJ/m

ol)

pH

Pi

4 5 6 7 8 9 10-50

-45

-40

-35

-30

-25

-20

(b) TFMSA

EZAbG

obs (

kJ/m

ol)

pH

5 6 7 8 9 10

-60

-40

-20

0

(a)

Tris

bH

ob

s (

kJ/m

ol)

pH

Pi

5 6 7 8 9 10-55

-50

-45

-40

-35

EZA

Method (buffer):

ITC (Pi)

ITC (Tris)

TSA

(b)

intrinsic

bG

(kJ/m

ol)

pH

obs

44

Binding and protonation

45/84

Observed vs Intrinsic Kd

5 6 7 8 9 1010

-11

10-10

10-9

10-8

10-7

IntrinsicIntrinsic

Hsp90-Rdc

Kd (

M-1)

pH

CA-EZA

5 6 7 8 9 10

(b)(a)

Observed

Observed

pH

46

Intrinsic EZA binding to hCA XIII

47

hCA2-EZA-L547C9 (active center)

r= 0.212069 free_r= 0.232668

Zn

His119A

His94A

Leu198A

Gln92A

48

CA II (greenish) – CA XIII (brownish)

49

VD compound series

1.E+04

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

1.E+11

VD10-35-2 VD10-39a VD11-4-2 VD11-16 VD11-31 VD11-33 VD11-39

hCA1-TS hCA2-TS hCA7-TS hCA12-TS hCA13-TS hCA14-TF

50

Some CA inhibitors selective or CA I

1,0E+04

1,0E+05

1,0E+06

1,0E+07

1,0E+08

1,0E+09

1,0E+10

TSA TSA ITC TSA ITC TSA ITC TSA ITC TSA ITC TSA TSA TSA ITC TSA

VD10-1

VD10-6 VD10-9 VD10-12 VD10-14 VD10-16 VD10-18

VD10-20

VD10-21A VD10-27

Ju

ng

imo

si

ko

ns

tan

ta

CA1

CA2

CA4

CA7

CA13

51

52

Intrinsic binding parameters, CA I

CA inhibitor publications

53

Conclusions

•It is important to distinguish observed from intrinsic binding parameters

•All binding and inhibition assays will provide only the observed thermodynamic parameters

•Only intrinsic parameters may be correlated with structure

•ICPD compounds are good anticancer leads

54