Recent applications of isotopic labeling for protein NMR in drug discovery

1. Introduction

2. Brief overview of

“protein-based” NMR methods

used in drug discovery

3. Biological systems used for

isotopic labeling

4. Advances in specific/selective

isotope labeling techniques

5. In-cell NMR: pharmaceutical

NMR applications for cellular

biochemistry and drug delivery

6. Expert opinion

Review

Recent applications of isotopiclabeling for protein NMR in drugdiscoveryHidekazu HiroakiNagoya University, Graduate School of Pharmaceutical Sciences, Nagoya, Japan

Introduction: Nuclear magnetic resonance (NMR) applications in drug discov-

ery are classified into two categories: ligand-based methods and protein-

based methods. The latter is based on the observation of the 1H-15N HSQC

spectra of a protein with and without lead compounds. However, in order to

take this strategy, isotopic labeling is an absolute necessity. Given that each1H-15N HSQC signal corresponds to a residue of the target protein, signal

changes provide specific information on whether a compound will fit into a

pocket. Thus, this protein-based method is particularly suitable for fragment-

based approaches, such as “SAR-by-NMR” and “fragment-growing.”

Alternatively, the information from a protein interface may be used to

develop inhibitors for protein--protein interactions.

Areas covered: This review discusses at the experimental procedures for

preparing isotopically labeled protein and introduces selected topics on

atom-specific and residue-selective isotope labeling, which may facilitate

the development of PPI/PA inhibitors. Furthermore, the author reviews the

recent applications of “in-cell” NMR spectroscopy, which is now considered

as an important tool in drug delivery research.

Expert opinion: Many recent advances in labeling methods have succeeded in

expanding NMR’s potential for drug discovery. In addition to those methods,

another new technique called “in-cell NMR” allows the observation of

protein--ligand interactions inside living cells. In other words, “in-cell NMR”

may become a pharmaceutical NMR technique for drug delivery.

Keywords: amino acid type-selective labeling, fragment-based approach, in-cell NMR, inverse

labeling, protein-based method, SAIL method

Expert Opin. Drug Discov. (2013) 8(5):523-536

1. Introduction

Nuclear magnetic resonance (NMR) spectroscopy has recently become an indis-pensable analytical method for drug discovery and drug design because it may pro-vide considerable information on molecular interactions as well as molecularstructures at the atomic level. The number of NMR application methods dedicatedto pharmaceutical sciences has increased drastically during the past decade due tothe advances in methods and increased sensitivity of spectrometers.

NMR applications in drug discovery are roughly classified into twocategories [1-4]. The first one is “ligand-based methods,” which include T1 andT2 differences, transferred-NOE [5], SHAPES strategy [6], DOSY [7], affinityNMR [8], STD [9], Water-LOGSY [10], ILOE [11], and INPHARMA [12]. All theseapproaches focus on the differences in the NMR signals derived from ligands, whichmake it unnecessary to use isotopically labeled target protein samples.

The second one is “protein-based methods” for which isotopic labeling is neces-sary. After introducing suitable stable isotopes (13C, 15N, or both), heteronuclearcorrelation spectra such as HSQC are measured. It is known that the NMR

10.1517/17460441.2013.779665 © 2013 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X 523All rights reserved: reproduction in whole or in part not permitted

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chemical shift is highly sensitive to the chemical and stericenvironment of an atom. HSQC spectra provide informationon whether a small molecule binds to a target protein andwhich residues in the target protein’s pocket interact withthe small molecule. This residue-specific or, sometimes,atomic level information can be efficiently utilized to furtherdrug design. Therefore, protein-based methods are used notonly for initial drug screening but also for validating the hitcompounds and subsequent step-by-step lead optimization.Another possible application is developing inhibitors of

protein--protein interactions (PPIs) and protein assemblies(PAs). In this post-genomic era, many intracellular proteinsin the human genome have been shown to participate incertain PPI networks, which are target candidates fornext-generation drug discovery. In addition, intrinsic fibrousproteins such as cytoskeletal components and membraneskeletons as well as pathogenically fiber-forming proteinssuch as a-synuclein and Ab(1-40/42) peptides should becometherapeutic targets for unmet medical needs. Because protein-based NMR methods provide considerable information onPPIs and PAs, advances in this area will realize a new horizonfor drug discovery of PPI/PA inhibitors.In this review, experimental procedures for preparing isoto-

pically labeled protein samples have been overviewed. Selectedtopics on atom-specific and residue-selective isotope labelinghave also been introduced, which may facilitate the develop-ment of PPI/PA inhibitors. In addition, recent applicationsof “in-cell” NMR spectroscopy, which is now considered asa new research tool in drug delivery, have been discussed.

2. Brief overview of “protein-based” NMRmethods used in drug discovery

2.1 Theoretical background and limitationsMost protein-based methods rely on two- or higher-dimensional NMR for heteronuclear chemical shift correla-tion. This is simply because 1H 1D NMR spectra of targetproteins are heavily overlapped thereby less informative.

This signal overlapping is solved by introduction of anotherdimension, either 15N or 13C. The natural abundance of15N and 13C are 0.4% and 1.1%, respectively. Thus, additionof other compounds does not interfere with the 1H-15N and1H-13C HSQC spectra of target proteins.

The methodologies for heteronuclear multidimensionalNMR of proteins were developed for high-resolutionstructural determinations. The typical steps in these studiesinclude the following: i) preparing a 15N-uniformly labeledprotein, ii) measuring 1H-15N HSQC, iii) making a go/no-godecision to continue the project, iv) choosing a labeling strat-egy, v) preparing either a 1H/15N/13C or 2H/15N/13C-labeledprotein based on the labeling strategy, vi) accumulating fulldatasets of 3D/4D NMR, vii) making sequential assignmentsfor backbone NH signals, viii) making nearly complete assign-ments for the side chain signals, ix) collecting NOEs and otherstructural restraints derived by NMR experiments as much aspossible, and x) calculating the solution structure that fulfillsall NMR-derived experimental data (Figure 1).

For modern drug discovery using NMR spectroscopy, theinformation obtained in step (vii) along with HSQC spectraof the target protein is used for its residue-specific sequentialassignments. The obtained 3D structure of the target proteinis also useful for further structure-guided drug design;however, this can be replaced with X-ray-derived orhomology-modeled structures. Hence, obtaining completesequential assignments of NH signals is not absolutely necessary.

Several limitations of these protein-based approaches shouldbe taken into consideration. First, any of the solution NMRtechniques are only applicable for relatively smaller proteins(i.e., molecular weights < 30 K). This limitation arises as aresult of the property of magnetization relaxation of slowlytumbling molecules, such as large proteins. Second, not onlythe theoretical molecular mass but also an increase in theapparent molecular mass of a protein may hamper the qualityof HSQC spectra. Nonspecific self-association of proteins isone of the major causes. Third, the presence of chemicalexchanges and equilibria between two or more states also ham-pers spectral quality. This may occur when a protein includesdynamic motions in its molecule. Fourth, NMR measure-ments are not highly sensitive, and a high concentrationprotein sample (~ 10 mg/ml) is required. For the latter threereasons, extensive optimization of a sample’s conditions isusually required; otherwise, informative and analyzable HSQCspectra cannot be obtained. In addition to these demerit, itshould be noted that the NMR-derived information ofdrug--protein interaction is rather qualitative than quantitative.Although Kd can be calculated with a saturation curve derivedfrom NMR titration experiments, it is only available forrelatively weak interaction such as sub-micromolar order.

2.2 Featured applications of the protein-based NMR

approach: NMR-assisted FBDDA trend in lead generation is the fragment-based approach.This approach is started to find one or several small molecular

Article highlights.

. Recent advances on HSQC-based NMR methods fordrug discovery as well as isotopic labeling methodsare reviewed.

. Not only Escherichia coli expression systems but alsoBrevibacillus, Kluyveromyces lactis, and Pichia pastoris,plant cells, insect cells, and mammalian cells are nowavailable for isotopic labeling.

. Amino acid-specific labeling, inverse labeling, andsegmental labeling techniques are used to simplify thespectra thereby focusing on protein pockets of interest.

. 13C-fractional labeling, alternating Ca labeling,methyl-specific labeling, and SAIL methods are used toexpand the limit for observing larger proteins.

This box summarizes key points contained in the article.

H. Hiroaki

524 Expert Opin. Drug Discov. (2013) 8(5)

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weight compounds called fragments that weakly bind to thebinding site of a target protein. Two of these are selectedand linked together with an appropriate linker moiety,resulting in a high-affinity drug candidate (Figure 2A).Alternatively, one fragment molecule is modified to growstep-by-step, gaining further affinity until all pocket vacanciesare filled (Figure 2B). In many cases, both strategies are usedin combination.

NMR-assisted FBDD is a concept in which the informa-tion derived from a target protein in the presence of acompound is fed back to design and synthesize the nextmolecule. One of the pioneering efforts of this concept is“SAR-by-NMR” that was proposed by Shuker et al.(Figure 2A) [13]. First, 1H-15N HSQC spectra were recorded.The resonances around the mutual drug-binding pockets Aand B should be uniquely identified. Subsequently, byNMR titrating experiments, the first ligand that binds topocket A is identified using the HSQC signals surroundingthe pocket as a guide. This molecule (ligand a) is modifiedto form ligand b with higher affinity. Subsequently, in thepresence of ligand b, a compound (ligand c) that binds topocket B is screened and optimized to form ligand d. Finally,ligands b and d are connected by an appropriate linker moi-ety. With this approach, NMR signals of backbone amidesare used to monitor how well the fragment molecules fitinto the cavities. The potential of this method was firstdemonstrated by Abott’s group in discovering a nonpeptidicstromelysin inhibitor [14]. Recently, the same group has

reported the application of this method for selectiveBcl-2 inhibitor [15]. The original method only describes theapplication using 1H-15N HSQC, although a similarexperiment using 1H-13C HSQC with a 13C-labeled proteinhas also been considered.

3. Biological systems used for isotopiclabeling

3.1 Escherichia coli expression systemsSimilar to many other biotechnology applications, E. coliprotein expression systems are the first choice for the practicalpreparation of isotopically labeled protein samples [16,17]. Oneof the widely used approaches is to use a plasmid that carriesan inducible T7 RNA polymerase promoter to express thegene of interest, which is subsequently used to transformBL21(DE3) E. coli cells [18]. Alternatively, ptac/plac-promoter-driven or pCold (cold shock protein) [19] promoter-drivenexpression vectors with appropriate purification tags (glutathi-one-S-transferase, maltose-binding protein, and thioredoxin)are used.

In all cases, NMR minimal media containing 2 -- 4 g/l ofglucose (12C6- or 13C6-) as the sole carbon source and0.5 -- 1 g/l of ammonium chloride (14N- or 15N-) as thesole nitrogen source and supplemented with some vitamins,minerals, and nucleosides are used as the harvesting medium.Because the metabolic pathways of E. coli have been almostcompletely analyzed, many variations of selective/specific

Signal assignments restraints

Distancesdihedralangles

go/no-godecision

3D/4DNMR

15Nsample

15N/13Csamples

9.0(1H) 8.0 7.0 (ppm)

(ppm)

125

120

115

110

(15N)

Figure 1. Schematic drawing of the standard solution NMR protocol for protein structure determination (see text). Usually,

the first step is to obtain uniformly 15N-labeled protein samples, and go/no-go decision is made according to the quality of1H-15N HSQC spectra as a guide. After analyses of several 3D/4D-NMR data, full assignments of back-bone amide proton

resonances are achieved. Further structural restraints (inter-proton distances and dihedral angles) are accumulated and

subsequently used for the structure calculation step.

Recent applications of isotopic labeling for protein NMR in drug discovery

Expert Opin. Drug Discov. (2013) 8(5) 525

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isotope labeling are now obtained using wild-type andgenetically modified E. coli strains (see Section 4).

3.2 Brevibacillus choshinensisA new prokaryotic protein-expressing host species has beenidentified. Brevibacillus choshinensis HPD31-SP3 is a gram-negative bacterium [20]. The origin of this strain was formerlyknown as Bacillus brevis, which was reclassified and renamedBrevibacillus. Related strains are well-known hosts for highlevel protein secretion and low extracellular proteaseactivity [21]. A B. choshinensis expression system was furtherdeveloped and fine-tuned by the Higeta Shoyu Co., a soysauce brewer in Choshi, Japan. Tanio et al. demonstratedthat 15N-labeled bFKBP12 was secreted into the medium [22]

and also recommended using a commercially available semi-synthetic medium (C. H. L. medium, Chlorella IndustryCo., Japan) rather than an M9-based medium because oflower protein production. The authors successfully expandedtheir method to amino acid type-specific (AATS) labeling [23].A remarkable feature of a Brevibacillus expression system liesin its protein secretion pathway. Protein samples that containdisulfide bonds may be available using this system.

3.3 Pichia pastoris and other yeastsBacterial protein expression systems have many advantages,such as the ease of molecular genetic analysis and plasmidhandling, faster growth rates, and lower media costs.Nevertheless, heterologous protein production often fails forseveral reasons, particularly when expressing eukaryotic andmammalian genes. This phenomenon can be explained bythe following reasons: i) differences in the architectures andtranslation speeds of ribosomes, ii) presence or absence ofsubcellular components, iii) differences in the numbersof molecular chaperones, and iv) the presence or absence ofactivity for post-translational modifications [24].So far, an economical expression system using eukaryotic

cells has been long-awaited. Yeast can be used for this pur-pose. For example, recently, Kamiya et al. demonstrated the

overproduction of 13C-labeled homogenous oligosaccharides,which may become another source for pharmaceuticalresearch [25]. In contrast to Saccharomyces cerevisiae, the mostbasic model organism, Schizosaccharomyces pombe appears tobe more suitable for heterologous protein production [26]. Nev-ertheless, to our best knowledge, no isotopic labeling applicationof Sc. pombe has been reported. Williopsis mrakii, a fermentingbudding yeast for Japanese sake, was used to express 15N-labeledWm toxin, although this protein was not heterologous [27].

A minimal medium for yeasts can be used to harvest cul-tures. Thus, 15N-labeling is cost-effective, whereas 13C-label-ing is possible, but expensive. This is because the yeastmedium contains 10 -- 20 times more glucose than theE. coli M9 medium.

Therefore, P. pastoris, a methyltrophic yeast, is one of thebest choices in this category. In structural biology, P. pastorisis widely used for the large-scale production of membraneproteins, such as GPCRs. Isotopic labeling of NMR samplesby Pichia has been developed and established [28]. A Pichiaexpression system may be used in combination with analcohol oxidase 1 (AOX1) promoter. Pichia can grow in amedium with NH4Cl as the sole nitrogen source, and a15N-labeled protein sample is easily obtained at a reasonablecost, although, a problem may arise when attempting toobtain 13C-labeled proteins. An optimized protocol withregard to lowering the medium cost has been proposed [29].

3.4 Kluyveromyces lactisKluyveromyces lactis is another yeast that was recently estab-lished as a host for protein expression studies [30,31]. K. lactisis a well-known yeast used in the industry for the commercialproduction of rennet. K. lactis is also known as a killer yeastbecause it secrets a toxin (g toxin) that kills other yeast speciessuch as S. cerevisiae. Sugiki et al. successfully utilized a K. lactisexpression system to produce isotopically labeled proteinsamples with an acceptably low cost, even for 13C-labeling.

One of the major advantages of a K. lactis expression systemcompared with that of P. pastoris is its promoter for

Pocket A Pocket B

Ligand c Ligand d

A. B.

Figure 2. The two major strategies of "protein-based" approaches of NMR spectroscopy in drug discovery: (A) SAR-by-NMR

method, (B) fragment-growing method. Open and closed circles represent each NH groups in the target protein, whose

signals are either insensitive or sensitive to ligand binding, respectively. Filled arrows indicate the direction of the growth of

the initial fragment (light gray) by step-by-step lead optimization processes.

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gene expression. The strong LAC4 promoter, which inducescontinuous protein production in the presence of galactose inthe culture medium, was used for this purpose. Moreover,Sugiki et al. successfully optimized the culture conditions toreduce the total amounts of 13C-galactose used as a carbonand energy source. The original protocol used 2% (20 g/l)galactose [32]. Sugiki et al. reduced this to 5 g/l of 13C6-glucoseby employing a well-designed fed-batch harvesting protocol. Asa result, the cost of isotope (13C/15N) labeling the proteins ofinterest was as low as 20% of that used for P. pastoris. Becausethis cost is in the range of 100 -- 200% of that with E. coli,the K. lactis expression system should become more popular.

3.5 Insect, plant, and mammalian cellsIn addition to yeast expression systems, other highereukaryotes are now available as hosts for heterologous geneexpression in terms of isotopic labeling. However, the problemsof the cost and availability of isotopically labeled media persist.For insect cells, the well-known Sf9 cells with a baculovirusexpression system, which are widely used in molecular biologystudies, were also employed to prepare NMR samples [33-35].Uniform labeling and AATS labeling were achieved.

With regard to plant cells, Ohki et al. established an expres-sion system using plant cells [36]. Tobacco BY-2 cells wereused to demonstrate the production of sufficient amounts ofproteins, including dihydrofolate reductase, chicken calmodu-lin, porcine protein kinase C-dependent protein phosphatase-1 inhibitor, and bovine pancreatic trypsin inhibitor (BPTI).In this method, only 15N-labeled proteins are available byusing K15NO3 and 15NH4

15NO3 as nitrogen sources. It isnotable that BY-2 cells successfully produce BPTI with correctdisulfide bonds (three bonds in this protein).

The use of mammalian cells should also be noted, since theiruse for standard molecular biology experiments is very popular.In terms of isotopically labeled preparation of biologics andimmunoglobulins, mammalian expression systems are especiallyimportant, because their glycosylation profile is crucial for thepharmacological function (reviewed in [37]). So far, Sastry et al.recently demonstrated the preparation of isotopically labeled,NMR-competent samples for the outer domain of theHIV-1 gp120 glycoprotein using an adenovirus expressionvector [38]. This system was optimized for use with an NMR-ready synthetic medium for mammalian cells, CGM-6000(Cambridge Isotope Laboratory, Ltd.). Under optimized con-ditions, HEK293 cells could produce > 40 mg/l of proteinswith an average of > 80% isotope incorporation. This applica-tion will open the door for NMR structural biology usingmammalian expression systems, in addition to the reasonablecost for labeling.

3.6 Cell-free systemsSeveral cell-free protein synthesis systems and many commer-cially available kits have been developed [39]. Theoretically,there should be no difference between protein expressionwith natural (no label) and isotopically labeled amino acids.

Thus, the only issue for isotopic labeling is the productionrate. E. coli cell-free systems and wheat germ cell-free systemsare known for their use in highly automated protein produc-tion in several structural genomics projects. Based on thedegree of purification of cell-free extracts, which include theentire machinery of protein production along with other met-abolic enzymes, some of these are limited in their use foramino acid-specific labeling. Special care should be taken toavoid metabolic scrambling when chemically modified aminoacids, such as SAIL amino acids, are used [40].

4. Advances in specific/selective isotopelabeling techniques

Strategies for specific and selective isotope labeling techniquesare briefly summarized in this section (Figure 3). In general,these labeling strategies are roughly grouped into two categoriesbased on their purposes. The first group includes AATS label-ing, inverse labeling (AATS unlabeling), combinatorial AATSlabeling, and segmental labeling. These strategies are used tosimplify the measured spectra by decreasing the numbers ofobservable signals. These simplifications may contribute to acertain rationale for confirming already-made assignments ofsignals, thus contributing to the assignment process itself.

The second group of strategies includes metabolic frac-tional labeling methods and a chemical method for certainspecific moieties. The stereo-array isotope labeling (SAIL)method is one of the most extreme uses of chemical synthesis.This group of methods is primarily used to challenge andexpand upon the molecular weight limit of NMR methodol-ogy by improving the relaxation pathways of certain spins inthe protein of interest.

4.1 AATS labelingSeveral expression systems have been used to demonstrateAATS labeling [23,33,35,41-43]. To accumulate the complete setof AATS labeling for all amino acids, the system usingE. coli required a set of auxotrophs [44]. However, for someamino acids that are located at the terminus of a metabolicpathway, such as His, Lys, Met, and Ala, AATS labelingwith a prototrophic host strain is highly attainable [45].When using cell-free protein synthesis systems, AATS labelingappears to be much easier than when using expression sys-tems. However, some cell-free systems still contain active met-abolic enzymes; therefore, special care should be taken toavoid isotope scrambling [46].

Obviously, the greatest merit of this approach is its simplic-ity (Figure 3B). The residues of only the labeled amino acidgive HSQC signals without doubt. Thus, the process ofsequential assignment becomes simpler and more reliable. Insome difficult cases, such as low sample solubility or weakself-association, the quality of 3D NMR spectra is notsufficient to reliably complete the sequential assignments ofbackbone signals. AATS labeling can resolve this issue.



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1H(amide) (ppm) 15

N (

pp

m)

15N A.

ALA

1H(amide) (ppm)

15N

(p

pm

)

15N B.

ALA

1H(amide) (ppm)

15N

(p

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)

15N C.

ALA

1H(amide) (ppm)

15N

(p

pm

)

15N

13VALC D.

1H(amide) (ppm)

15N

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)

15NE.

1H(methyl - ppm)

13C

(p

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13C F.

1H(methyl - ppm)

13C

(p

pm

)

13C G.

M

M

M

M M

M

M

1H(methyl - ppm)

13C

(p

pm

)

13C / 2H H.

Figure 3. Schematic diagram for uniform and specific isotope labeling of a protein with its appearance in 2D NMR spectra.

Filled and double circles represent fully isotopically labeled amino acid residues with the indicate nuclei. Open and shaded

circles represent unlabeled and fractionally labeled amino acids, respectively. Circles with "M" denote residues with 13C-

methyl-specific labeling. (A) Uniform 15N-labeling, (B) 15N-AATS labeling (15N-Ala), (C) 14N-inverse-labeling (14N-Ala) in 15N

background, (D) combinatorial AATS labeling with 13C-Val and 15N-Ala. Only the signal corresponding to Val-Ala sequence

gives a doublet signal, (E) segmental labeling, (F) uniform 13C-labeling and the expanded CH3 region of 13C-HSQC spectrum.

Signals are broadened by 13C--13C coupling, (G) 13C-fractional labeling. proR methyl groups give doublet signals whereas proS

methyl groups are in singlet form, (H) 13C-methyl-specific labeling in 2H background. Signals are much sharper than (F).

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When combining a genetic strategy to prepare all Alasubstitution mutants for the target amino acid in the proteinof interest, the specific assignments for all certain aminoacid residues are possible without using any three- or higher-dimensional NMR techniques. Thus, the molecular weightlimitation of NMR methodology is also largely expanded(up to 100 K).

4.2 Amino acid-selective unlabeling (inverse labeling)Another approach is amino acid-selective labeling and unlab-eling. In this strategy, few 14N-amino acids are added to themedium with a 15N-background (Figure 3C). This methodwas originally developed by Shortle for simplifying HSQCspectra and for identifying amino acid residue types to aidin the subsequent sequential assignment process [47]. Werecently improved this protocol by combinatorial aminoacid inverse labeling without using auxotrophic E. coli hoststrains [48]. This method was optimized to obtain eight com-binations of amino acids, Ile/Leu/Val(-), Gly/Ser/Cys(-),Phe/Tyr(-), Ala(-), Lys(-), Arg(-), His(-), and Trp(-), whichwere inversely labeled in the standard E. coli host strainBL21(DE3) using a minimal M9 medium formulation.This technique was further expanded to facilitate sequentialassignments in a cost-effective manner [49].

4.3 Combinatorial AATS labeling for sequential

assignmentsAn advanced option for AATS labeling is simultaneouslyusing two or more differently labeled amino acids. For exam-ple, a combination of 13C-labeled Val and 15N-labeled Alaprovides unique 13C-coupled-15N signals only at the aminoacid sequence of Val-Ala (Figure 3D). Thus, combinatorial13C and 15N AATS labeling can be used for sequence specificassignments for specific amino acid pairs.

Kohno recently developed a method to produce dual aminoacid-selective 13C-15N labeled proteins by using an improvedwheat germ cell-free system [50]. This method opened thedoor to completing sequence-specific assignments of amidesignals for very large protein samples.

4.4 Segmental labelingSegmental labeling is another approach for simplifying thecomplex HSQC spectra of larger or multidomain proteins(Figure 3E) [51]. This method utilizes in vitro protein ligationof two individually prepared protein segments; one is isotopi-cally labeled, and the other is unlabeled. For protein ligation,the “split intein” approach is commonly used [52].

One of the major drawbacks of this strategy is that refold-ing of the ligated protein sample is absolutely necessary.Thus, applications for some fragile samples such as kinasesand other biologically important enzymes remain limited.A recent application of segmental labeling was optimized forimproving the solubility of smaller soluble protein samples [53].An appropriate fusion tag protein efficiently improves the

solubility of the protein of interest, which enables obtainingan NMR sample of sufficient concentration.

4.5 Metabolic fractional labeling and moiety-specific

labelingMetabolic fractional labeling is a method to achievestereo- and region-specific isotopic labeling of proteins usingisotopically enriched precursors of amino acids for biosyn-thetic pathways. For example, mixtures of 13C6- and12C6-glucoses (Figure 3G) [54], [1]-13C1-glucose [55,56], as wellas [1,3]13C2- and [2]13C2-glycerols [57,58] are in this category.Therefore, an E. coli expression system is often used with thesemethods. To the best of my knowledge, the first applicationof metabolic fractional labeling was the use of 13 -- 87%13C6- and 12C6-glucose mixtures for stereo-specific NMRassignments of prochiral methyl groups in Leu and Val resi-dues (Figures 3G, 4A) [54]. In addition, this approach is usedfor analyzing amino acid metabolic pathways in E. coli [59,60]

and successfully applied to backbone assignments [61].Alternatively, a method to selectively introduce 13CH3

groups of Val, Ile, and Leu within an otherwise fully2H-labeled background was developed (Figure 3H) [41,62]. Inthis case, 13C2-a-keto-b-methylvaleric acid was added to theE. coli culture medium. The isolated spin system of 13CH3

groups showed improved relaxation properties. A pulsesequence “methyl-TROSY” is employed to observe these13CH3 signals in very large proteins. In addition, this methodis suitable for protein--protein interaction interfaces in largerprotein complexes.

Recently, Takeuchi et al. demonstrated a method for alter-nating 13C labeling using either [1,3]13C2- or [2]

13C-glycerolas the carbon source in 15N/2H2O medium [58]. This methodprovided a protein sample in which a 13Ca atom was placedas the neighbor of 12C¢ and 12Cb; thus, their spin was isolated(Figure 4B). This method was effectively combined with13C-directly detected 13C-15N-2D NMR experiments(CaN). The prolonged relaxation properties of the isolated13Ca in larger proteins were advantageous for further NMRapplications, such as CACA-TOCSY experiments [63].

4.6 Chemical 13C-Me-Lys labelingChemically introducing 13C-labeled methyl groups ontoe-amino groups of lysine residues in unlabeled proteinsshould be noted [64]. Chemical methylation of lysines has along history for improving the resolution during X-raycrystallization [65] and was recently re-evaluated forhigh-throughput structural genomics [66,67]. So, this methodhas been expanded to protein NMR. In this case, a treatmentcalled “reductive methylation” is used.

In brief, 13C-formaldehyde is mixed with a target protein ina buffer at physiological pH, after which sodium cyanoboro-hydride is added to reduce the Schiff base complexes offormaldehyde and lysine. This reaction is so mild that itprovides the following advantages: i) methylation of lysine



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residues does not significantly change a protein’s structureduring and after the reaction, ii) the methylated lysines retaintheir positive charges, iii) this method allows the introductionof NMR-active nuclei into proteins purified from organismsfor which there are no available isotope enrichment protocolspost-translation, and iv) the 13C methyl groups on lysines

have favorable relaxation properties, which makes itapplicable for large molecular weight proteins.

4.7 SAIL methodTo further improve the relaxation properties of the atoms ofamino acids, SAIL adopted a strategy of an extreme use ofchemical synthesis [40]. This method employs stereo- andregion-specific patterns of stable isotopes in which all2H- and 13C-labeling sites in uniformly 15N-labeled proteinsare extensively designed. Their occupancy level is nearlycomplete (~ 100%). Selected examples of SAIL amino acidsare illustrated in Figure 4C.

The concept for designing the labeling patterns of SAILamino acids is as follows: i) one of the two protons at CH2

is substituted to 2H (D) resulting in 13CHD; ii) two of theprotons at the Ala, Thr, and Met methyl groups are substi-tuted to 2H (D) resulting in 13CD2H; iii) one of the prochiralmethyl groups of Val and Leu are also labeled with 13CD2H;iv) the other prochiral methyl site is labeled with 12CD3;v) finally, for the six-membered aromatic rings of Phe andTyr, 13CH and 12CD moieties are applied at alternating posi-tions. As a result, all 13C atoms possess single 1H whose NMRsignals are not hampered by geminal 2JHH coupling as well asrelaxation via 1H--1H and 1H--13C dipolar couplings.

Typically, these SAIL amino acids are introduced intoproteins through cell-free protein synthesis [68-70]. This methodprovides an opportunity to determine the structures of largemolecular weight proteins with a reasonable effort [71,72].

5. In-cell NMR: pharmaceutical NMRapplications for cellular biochemistry anddrug delivery

5.1 In-cell NMR in bacterial expression systemsAs mentioned previously, heteronuclear correlation spectros-copy for 1H/15N and 1H/13C is merely observable withoutthe aid of isotopic labeling due to their low naturalabundance. This is the greatest advantage of HSQC-based/protein-based NMR methods used in drug screening becauseonly the signals from the target protein of interest are visible,even in a highly heterogeneous sample with numerousimpurities. One of the most extreme examples of this isin-cell NMR spectroscopy (reviewed by [73,74]).

The first example of in-cell NMR in living E. coli cells wasprovided by Serber et al. [75,76]. The principle of introducingstable isotopes into a protein of interest for in-cell NMR isvery similar to the protocol used for standard solution NMRby overexpressing an 15N-labeled protein sample using aT7-promoter-based system. The only major difference isoptimizing the timing for switching the culture mediumfrom nonlabeled to an isotopically enriched medium justbefore isopropyl-b-thioglucoside (IPTG) induction of targetprotein synthesis.

Because the efficiency of accumulation of the protein ofinterest is sufficiently high, it is well known that even a crude

13C6-glucose 12C6-glucose

Val

Leu

13% + 87 %

β

β

Val Ile Leu

Trp

A.

B.

C.

Figure 4. Examples of metabolic and chemical atom-specific

isotope labeling of amino acids. In the scheme, *C denotes13C atom. (A) 13C-fractional labeling using mixture of13C6- and 12C6-glucose by E. coli metabolic pathways. The

signal-splitting pattern can be used for stereo-specific signal

assignment of methyl groups, (B) 13C-alternating labeling

with [1,3]-13C2- and [2]-13C1-glycerol. (C) Selected examples

of SAIL amino acids.

H. Hiroaki


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lysate of E. coli can provide a sufficient quality HSQCspectrum [16,77]. Thus, this technique has been expanded tomeasure a slurry sample of living bacterial cells that mayhave the overexpressed, isotopically labeled proteins. Becausethis methodology appears to be adequately robust, the bacte-rial in-cell NMR technique can be combined with many ofthe isotopic labeling techniques using E. coli cells describedin Section 4.

One of the most significant recent advances in bacterialin-cell NMR methods is the de novo NMR structure determi-nation of a protein from scratch. Sakakibara et al. successfullydetermined the structure of protein TTHA1718, a putativemetal binding protein from Thermus thermophilus, in livingE. coli cells [78]. The high viscosity and substantial instabilityof an in-cell NMR sample interfered with the complete resultsof a series of three-dimensional NMR spectra. They overcamethis problem by extensively using nonuniform samplingprofiles of multidimensional NMR spectra coupled withmaximum-entropy reconstruction rather than the usualFourier transformation [78,79].

Another remarkable application of bacterial in-cell NMRfor drug discovery is small molecule inhibitor library byin-cell NMR (SMILI-NMR) [80]. Before developing this,Shekhtman et al. developed a new variant of bacterial in-cellNMR method called STINT-NMR [81,82]. E. coli cellsharboring two different genes under the control of twodifferent promoters were used.

The first gene was allowed to be expressed in an isotope-labeled medium, after which the second gene was expressedin nonlabeled conditions. As a result, the first protein was vis-ible on NMR, whereas the second protein, which interactedwith the first protein, was not observable. Complex forma-tions of these two proteins could be monitored similar toin vitro protein--protein interaction experiments. This idea(SMILI-NMR) is applicable to examine the effects ofsmall molecules that potentially inhibit a protein--proteininteraction of interest [80].

5.2 In-cell NMR in Xenopus oocytesAnother application of in-cell NMR was successfully devel-oped using a Xenopus laevas oocyte system [83-85]. Xenopusoocytes are frequently used in drug discovery, particularlyfor the physiology of ion channels [86] and water channels [87].For these purposes, either mRNA or cDNA of the gene ofinterest is microinjected into oocytes, which then express thegene product. An automated, high-throughput system fordrug screening using Xenopus oocytes is now widely used [88].

Sakai et al. successfully made the first application of “in-cell NMR” for eukaryotic cells. 15N-uniformly labeled Ub(prepared with a standard E. coli expression system) wasmicroinjected into 200--250 oocytes at developmental stagesV--VI. Although up to 100 mM, 15N-labeled Ub accumula-tion in living oocytes caused severe signal losses. When com-paring wild-type and interaction-deficient Ub mutants, weconcluded that this signal loss was due to protein--protein

interactions inside the living oocytes [83,89]. We also demon-strated the monitoring of endogeneous enzyme activity of ade-ubiquinating protease that processed the C-terminalamino acid of 15N-Ub.

The application of Xenopus in-cell NMR for monitoringprotein--protein interactions was expanded to the study ofthe microtubule binding protein Tau [84]. Tau is an intrinsi-cally disordered, aggregation-prone protein that is stronglyassociated with neuronal degenerative diseases (tauopathiesand Alzheimer’s disease). The formation of cellular aggregatesof Tau, known as paired helical filaments (PHFs), and itssubsequent drop-out from neuronal microtubules is believedto underlie this molecular pathogenesis. Thus, the in vivobehavior of Tau is of special interest.

Alternatively, Hansel et al. established how to observeDNA and RNA NMR spectra inside living oocytes [85]. Thecritical point was chemically modifying natural nucleic acidsto avoid in vivo degradation.

All these papers have clearly shown the potential of in-cellNMR methodologies, which are applicable to studies indrug delivery.

5.3 In-cell NMR in mammalian cellsWith regard to its potential for evaluating drug deliverysystems, in-cell NMR using mammalian cells has been long-awaited. To date, two applications have been published [90,91];one of these applications was proposed by us. In both meth-ods, the difficulty was introducing isotopically labeled proteinsamples into living cells. In contrast to bacterial systems, anefficient, rapid protein expression system for the genestransfected into mammalian cells has not yet been established.Further, in contrast to Xenopus oocytes, microinjecting labeledproteins into mammalian cells in large amounts isnot realistic.

Thus, Inomata et al. overcame this problem by using atechnique called a protein transduction domain (PTD) tag.PTD, also known as a cationic membrane penetrating peptide(CPP), has emerged in applications for drug delivery ofmacromolecules and biomedicines, such as bioactive peptides,antibody drugs, and antisense oligonucleotides [92-94]. Theefficiency of protein transduction is boosted in combinationwith pyrenebutyrate [95]. HeLa and COS7 cells were shownto be competent for in-cell NMR methods in which 15N-labeled Ub, FKBP12, and GB1 were observed by NMR [90].Further, we succeeded in monitoring signal changes ofFKBP12 in the presence and absence of a binding drug,FK-506. This was the first biophysical evidence ofprotein--drug interactions at the atomic level in living cells.

Another means to deliver 15N-labeled thymosin b4 (Tb4)into 293F cells was demonstrated by Ogino et al. They useda pore-forming bacterial toxin, streptolysin O (SLO). SLOproduces pores with a diameter of 25 nm on the cell mem-brane, which are then resealed in a medium that contains Ca2+. Using this technique, they transduced Tb4 and successfullyobserved its HSQC spectra. In addition, post-translational



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N-terminal acetylation of Tb4, which resulted in certainchemical shift changes, was observed.In conclusion, although mammalian in-cell NMR method-

ologies have not yet matured and are still under development,their potential for pharmaceutical applications is noteworthy.

6. Expert opinion

In drug discovery research, solution NMR spectroscopy withboth isotopically labeled and unlabeled proteins has enormouspotential for target protein validation, initial drug screening,hit evaluation, lead optimization, and even for ADMEimprovement. In this review, I did not focus on ligand-basedmethodologies. However, the increasing number of newapplications and techniques with ligand-based approachesmust not be negligible. In addition, both strategies, protein-based and ligand-based methods, are highly complementaryto each other. Thus, researchers must attempt to establish anexpression system for their target protein of interest to incor-porate isotopes and to increase the number of applications ofHSQC-based experiments.For example, not only chemical shift perturbation experi-

ments (i.e., SAR-by-NMR) but also many other physicaland physicochemical parameters such as relaxation times,H/D-exchanging rates, conformational exchanges on millisec-ond time scales, and residual dipolar couplings are nowbecoming accessible by introducing isotope labels. Thisfine-tuned information at individual residue or atomic levelresolution is suitable for characterizing relatively difficultdrug targets.For example, modifying and controlling PPIs and PAs by

low molecular weight compounds are attractive concepts fornext generation drugs. In such cases, X-ray crystallographymay not be efficient because protein complexes, fibrils, andaggregates are sometimes rather transient and heterologous,which precludes their crystallization. In the same context,intrinsically disordered proteins (IDPs) are also importanttargets that can be studied only by solution methods.The use of in-cell NMR spectroscopy for basic research on

drug delivery is another possible option. Currently, in-cellNMR is the only way to monitor a protein at work inside liv-ing organisms, which may provide new insights in the mech-anism of drug action. However, the very low signal-to-noiseratios of proteins inside cells hinder the rapid spread of thistechnology. In particular, for mammalian applications of in-cell NMR, there have been only two successful examples.

The method used for incorporating labeled proteins insideliving cells at a high concentration is the difficulty. Othermethods, such as overexpression of labeled proteins insideliving mammalian cells must be developed.

A recent advancement in the high-throughput, automatedstructure determinations of proteins provides another per-spective. This topic may only attract the attention of research-ers within the field of structural genomics. However, decadesafter the initiation of structural genomics research projects,two consortia (in Europe and the US, but not in Japan) con-tinue to develop and refine the pipeline for structure determi-nations by NMR [96,97]. A highly automated pipeline ofprotein structure determinations may become readily applica-ble to drug design.

In case of structure-guided drug design, the speed ofobtaining a structure of a protein--drug (candidate) complexis the rate-limiting factor. A part of this problem has alreadybeen solved by high-throughput X-ray crystallography ofdrug--target complexes. Some pharmaceutical companieshave their own synchrotron beam lines. The remainder ofthis problem will be solved soon by high-throughput NMRstructural determinations. This will be possible by combiningknown techniques to accelerate NMR experiments includingnonuniform sampling and reduced dimensionality techniquesfor rapid measurements of 3D/4D-spectra, SOFAST-HMQC- and BEST-HNCA-based pulse sequences also usedfor shortening data acquisition times, and an automatedprotocol for signal assignments from NMR data [98,99]. Theseapproaches can be used in place of high-throughput X-raycrystallography.

With the help of high-performance computing andaccumulation of a large number of examples of protein--drugcomplexes, it should not be surprising that the paradigmsused in rational drug design and discovery will change duringthe next five years.

Acknowledgments

The author thanks K Sugiki and H Takahashi for helpfuladvice. The author also thanks Enago (www.enago.jp) forthe English language review.

Declaration of interest

The author declares no conflict of interest and has received nopayment in preparation of this manuscript.

H. Hiroaki


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http://www.enago.jp

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AffiliationHidekazu Hiroaki PhD

Nagoya University,

Graduate School of Pharmaceutical Sciences,

Furocho, Chikusa-kum, Koto-kenkyu-kan,

Nagoya, 464-8601, Japan

E-mail: [email protected]

H. Hiroaki


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Documents

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