Strategies for the Discovery of Target-Specific or Isoform-Selective

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Strategies for the Discovery of Target-Specific or Isoform-Selective Modulators Peng Zhan,*,† Yukihiro Itoh,‡ Takayoshi Suzuki,*,‡,§ and Xinyong Liu*,† †

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, 250012 Jinan, Shandong, P. R. China ‡ Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-Cho, Sakyo-Ku, Kyoto 606-0823, Japan § CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ABSTRACT: Currently, the creation of class- and isoformselective modulators of biologically important targets is a particularly challenging problem because different isoforms within a protein family often show striking similarity in spatial quaternary structure, especially at the catalytic sites or binding pockets. Therefore, an understanding of both the precise threedimensional structure of the target protein and the mechanisms of action of modulators is important for developing more effective and selective agents. In this Perspective, we discuss currently available rational design strategies for obtaining class- and isoform-selective inhibitors and we illustrate these strategies with the aid of specific examples from the recent literature. The strategies covered include: (1) target-derived (-dependent) de novo drug discovery methodologies, and (2) follow-on derivatization approaches from initially identified active molecules (hit-to-lead and lead-to-candidate efforts). We also comment on prospects for further development and integration of strategies to achieve target-specific or isoform-selective inhibition.

1. INTRODUCTION The members of families of multifunctional enzymes, including epigenetic enzymes such as histone deacetylases (HDACs) and protein kinases such as phosphoinositide-3 kinases (PI3Ks), tankyrases (TNKSs), and nitric oxide synthases (NOSs), often have extraordinarily diverse cellular functions and are therefore compelling biotargets for the treatment of serious diseases such as cancer, diabetes, immune and inflammatory disorders, cardiovascular disease, and neurodegeneration.1−7 The stateof-the-art in structural and biochemical investigations of these multi-isoform enzyme families, as well as the family architectures, has been well reviewed elsewhere.1−7 However, the results of clinical trials of chemical modulators of specific enzyme targets within these families have often been disappointing because of effects such as insufficiently specific inhibition, off-target activities, and toxicity. Insufficient selectivity is thus one of the major bottlenecks in new drug development, although there are also other issues, including limited efficacy and the emergence of acquired drug resistance. Therefore, it is extremely important to discover potent inhibitors with strict subtype selectivity, not only as candidate therapeutic drugs but also as chemical tools to elucidate the roles of specific isoforms in human diseases and for mechanistic studies at the molecular level. However, it is tremendously challenging to identify modulators that are selective for one isoform of a large protein family containing homologous active © XXXX American Chemical Society

sites; for example, the kinase family contains more than 500 isozymes, which generally have highly conserved ATP catalytic sites. It is not too much to say that the discovery of selective isoform inhibitors is currently at the heart of medicinal chemistry and chemical biology.8 Thus, to address the formidable challenge presented by the specific molecular recognition of single protein targets within complex, multicomponent biological systems, we require highly sophisticated drug design strategies.8 Early discoveries of target-specific or isoform-selective agents were usually made by means of trial-and-error approaches, but these are time-consuming and unreliable. Therefore, there is increasing interest in rational design strategies. In this Perspective, we provide an overview of currently available design strategies to obtain selective modulators (Figure 1), with illustrative examples. Consequently, the many selective inhibitors discovered serentipitously or by trial-and-error are not included in this Perspective. Received: February 9, 2015

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DOI: 10.1021/acs.jmedchem.5b00229 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Schematic overview of medicinal−chemical strategies for discovery of target-specific or isoform-selective inhibitors.

Figure 2. Structure of inhibitor 1 (colored in yellow) complexed with nNOS (heme domain) (PDB code: 3B3N).

2. TARGET-DEPENDENT DE NOVO DISCOVERY OF TARGET-SPECIFIC OR ISOFORM-SELECTIVE INHIBITORS Recent advances in the understanding of protein−ligand interactions and structural bioinformatics of molecular recognition have encouraged rapid discovery of potent and selective molecules for therapeutically relevant targets. Over the past decade, a variety of strategies and techniques have been developed to facilitate de novo design of target-specific or isozyme-selective inhibitors with high potential for further modification. 2.1. Structure-Based de Novo Design. The structurebased approach is intrinsically limited owing to the inherent structural flexibility of proteins. Moreover, the active site pockets of isozymes are usually highly conserved, making the structure-based rational design of the isoform-selective inhibitor even more challenging. Nitric oxide synthase (NOS) represents a highly druggable family of biotargets that have received great attention in recent years. Structure-based drug design has played a crucial role in the identification of isoform-selective NOS inhibitors. Starting

with previously developed nitroarginine-containing dipeptides, novel selective neuronal nitric oxide synthase (nNOS) inhibitors have been created by using combinations of structure-based drug design, minimal pharmacophoric elements, and the fragment-hopping concept.9,10 The fundamental idea of this strategy is derivatization of a basic pharmacophoric component, taking into account the structures of the sites determining ligand binding and isoform selectivity. For the nNOS studies, a basic fragment library, a bioisostere library, a side chain library, a toxicophore library, and a set of rules for metabolic stability were employed to construct focused fragment libraries and then the fragments were connected. To meet hydrophobicity/steric constraint requirements, the 2aminopyridine motif was selected as a basic core. Finally, a small molecule 1 (Figure 2) with a new chemical scaffold was identified.10 Compound 1 showed nNOS-inhibitory activity at nanomolar concentration, with more than 1000-fold selectivity for nNOS over other NOS isoforms. Interestingly, the selectivity of dipeptide/peptidomimetic binders for nNOS over endothelial nitric oxide synthase (eNOS) was investigated by crystallographic analysis, and the results indicated that a B



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Figure 3. Proposed mechanisms of (a) LSD1 catalysis and (b) activation of 2, leading to inactivation of LSD1 by flavin modification.

Figure 4. (a) Crystallographic overlays of reduced FAD−N-propargyl lysine peptide conjugate (PDB code: 2UXN) (green and blue) and FAD− PCPA conjugate (PDB code: 2UXX) (magenta) in the catalytic site of LSD1. For the sake of clarity, amino acid residues in the catalytic site are omitted. (b) Chemical structures and in vitro inhibitory activities of compounds 3−5 toward LSD1 and MAO-A/B.

protein, the type of enzyme-catalyzed reaction, the size and shape of the catalytic site, and the availability of suitable reactive functional moieties or cofactors. 2.2. Mechanism-Based Drug Design. A detailed understanding of the interaction between the target protein and its ligands provides a good basis for improving the selectivity and

small-molecular compound with a constrained conformation, such as 1, could exactly mimic the mode of action of the dipeptide inhibitors of nNOS. Unlike the traditional de novo approach, which may not encompass the whole biologically relevant chemical space of the target enzyme, selection of the scaffold in the new approach is based on the nature of the target C



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Figure 5. Discovery of mechanism-based LSD1-selective inactivators.

Figure 6. Identification of carbonic acid isozyme-selective binders from a dynamic constitutional library. Target-driven self-screening of inhibitors occurs until the system reaches a thermodynamic minimum.

sion of LSD1 activity.13,14 Mass spectrometric analysis showed that 2 interacts covalently with FAD (Figure 3b). Since then, it has been used as a lead compound for anticancer drug discovery and as a tool for probing the mechanism and biological functions of LSD1. Compound 3 (phenylcyclopropylamine, PCPA), which is an inhibitor of monoamine oxidases (MAOs; also FAD-dependent enzymes, like LSD1), was also identified as a weak and nonselective LSD1 inhibitor that binds directly to the catalytic site.12 Superposition of the FAD−PCPA conjugate (PDB code:

affinity of inhibitors through mechanism-based drug design. This approach is well illustrated by the discovery of lysinespecific demethylase 1 (LSD1, or KDM1A)-selective inhibitors.11,12 LSD1 is a flavin-dependent amine oxidase that catalyzes oxidative removal of one or two methyl group(s) from the methyl-lysine-4 side chain of histone H3 (H3K4) (Figure 3a). In 2006, peptide-based compound (2) containing a propargylamine functionality was identified as a selective, mechanismbased inactivator of LSD1, exhibiting time-dependent suppresD



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Figure 7. Target-guided assembly of selective binders via azide−alkyne 1,3-dipolar cycloaddition.

Figure 8. Discovery of HDAC8 inhibitor (12) via protein-templated click chemistry.

technique has been employed to selectively assemble ligands with optimum molecular fit around a suitable target from a mixture of all possible combinations of the available components under thermodynamic control by taking advantage of structural information. This target-guided synthetic methodology (TGS) enables efficient generation of novel smallmolecular ligands for drug discovery, i.e., selective inhibitors can be identified by screening a complex compound mixture in the presence of the selected protein template.17,18 Since the introduction of this technique in the mid-1990s, many reversible covalent reactions have been used in the construction of dynamic constitutional libraries (DCLs), including reactions of imines, hydrazones, and disulfides. Recently, a DCL targeting carbonic anhydrase isozyme-specific inhibitors was developed.19 As shown in Figure 6, the DCL was assembled under thermodynamic control by using reversible amino-carbonyl/imine reaction and screened for binders to the metalloenzyme carbonic anhydrase (CA). There are 15 human (h) CA isoforms, and in this work, the DCL was screened in parallel for binders to hCAI and hCA II (Figure 7).19 When hCAI or hCA II is added to the DCL, the composition of the DCL (9a, 9b, 10a, 10b, 11a, 11b) adjusts to minimize the free energy of the overall system, resulting in amplification of selective ligands of the target protein. In situ click chemistry is another TGS method for fast and efficient production of potential enzymatic inhibitors. A wellknown example is the assembly of complementary alkyne and azide building blocks at binding sites of the target via a Huisgen

2UXX) and the reduced FAD−N-propargyl lysine peptide conjugate (PDB code: 2UXN) on the active site of LSD1 (Figure 4) led to the discovery of novel small-molecular LSD1selective inhibitors 4 and 5.15 Notably, compounds 4 and 5 showed inhibition of in vivo H3K4-methylating activity and antiproliferative potency against cancer cells. It can be anticipated that the similarity in the active sites and structural properties of LSD1 and MAO will prompt investigation of more anti-MAO agents as potential selective LSD1 inhibitors. On the basis of this concept of LSD1-targeted PCPA delivery, a series of novel LSD1 inhibitors was designed and synthesized. First, it was confirmed that peptide (6) inhibits LSD1 efficiently and selectively by delivering PCPA directly to the LSD1 active site, i.e., the inhibition occurs by formation of the FAD−PCPA conjugate with concomitant release of the peptide carrier (7).16 Then, the same strategy was utilized to develop nonpeptide small-molecular LSD1 inhibitors (Figure 5). These molecules showed potent LSD1-selective inhibitory activity via LSD1-targeted PCPA delivery in enzymatic assays, and they also displayed potent growth-inhibitory activity toward cancer cell lines. Among them, compound 8 is a potent LSD1 inhibitor (IC50 = 0.30 μM), being at least 100-fold more active than PCPA itself (IC50 = 31 μM) without inhibiting MAO-A (IC50 > 25 μM). Thus, compound 8 is a promising candidate for hit-to-lead follow-up in the anticancer drug discovery process. 2.3. Target-Templated Dynamic Combinatorial Chemistry. The dynamic combinatorial/covalent chemistry (DCC) E



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alkyne−azide 1,3-dipolar cycloaddition reaction, as illustrated in Figure 7. This strategy can be divided into three stages: (i) small building blocks do not react with each other in the absence of a target protein but bind to adjacent subsites, (ii) the proximity of alkyne and azide groups is conducive to 1,3-dipolar cycloaddition, and (iii) the resulting triazole displays very high binding affinity with the target protein. In 2012, in situ click chemistry of alkyne and azide building blocks on acetylcholinebinding protein templates (AChBPs; structural mimics of the extracellular domain of nicotinic acetylcholine receptors) afforded a series of triazole-based selective agonists for the α7 nicotinic acetylcholine receptor.20 In 2010, it was reported that a CuI complex of HDAC8 could accelerate selective azide−alkyne cycloaddition reaction, and HDAC inhibitor (12) was identified as a more potent inhibitor than either of its component parts alone (Figure 8). This example of in situ inhibitor formation provided a precedent for applying in situ click chemistry to obtain inhibitors of highly active protein-based catalysts.21 2.4. Virtual Screening. In recent years, structure-based virtual screening (usually involving a combination of structurebased drug design and computational docking) has become an attractive alternative to traditional high-throughput screening (HTS) for early stage hit discovery in both the pharmaceutical industry and academia. Although it relies heavily on knowledge of the three-dimensional structure of the target protein, it is an effective, low-cost, labor-saving strategy for drug discovery.22−24 In 2012, a series of selective aldo-keto reductase AKR1C1 and AKR1C3 inhibitors with novel scaffolds (exemplified by 13, Figure 9) was discovered through virtual HTS of a fragment library, followed by bioactivity assays with human aldo-keto reductases 1C1−1C4 (AKR1C1−AKR1C4).25

Figure 10. Crystal structure of compound (+)-14 complexed with human BChE (PDB code: 4TPK).

subfamily enzyme members. Availability of detailed structural information for whole enzyme families in the future will be extremely useful in the design of selective inhibitors for individual isoforms.

3. DISCOVERY OF TARGET-SPECIFIC OR ISOFORM-SELECTIVE INHIBITORS VIA DERIVATIZATION APPROACHES 3.1. Conversion of Substrates to Selective Inhibitors. Fragment-based drug discovery (FBDD) has emerged as a powerful strategy for drug discovery starting from simple substrate fragments.28 This strategy involves screening a relatively small library of substrate derivatives to identify novel scaffold leads for subsequent iterative optimization of potency and selectivity. Development of isoform-selective epigenetic modulators is of great biological and medical interest, and the substrate-based fragment derivatization approach has been applied to the development of small-molecular selective inhibitors of histone deacetylase (HDAC), sirtuin (SIRT), and Jumonji C domaincontaining histone demethylases (JHDMs) (Figure 11).29−34 In enzyme-inhibitory assays using HDAC1, HDAC4, and HDAC6, compounds 17−19, which were derived from HDAC6-selective substrate 15, showed selective inhibitory activity for HDAC6 over HDAC1 and HDAC4.29,30 As regards SIRT inhibitors, compound 21, bearing an ethoxycarbonyl moiety at the α-position to the acetamide of acetylated lysine substrate analogue 20, showed potent activity in an in vitro recombinant SIRT1 inhibitory assay, with high selectivity over SIRT2 and SIRT3. Mechanistic studies through kinetic analysis, mass spectroscopy (MS), and computation revealed that the enol form of 21 nucleophilically attacks NAD+ in the SIRT active site to form the stable 21−ADP-ribose adduct, resulting in potent enzyme inhibition. Compound 21 induced a dose-dependent increase of p53 acetylation in human HCT116 cells (colon cancer), indicating that it inhibits SIRT1 at the cellular level.31 Inspired by the binding modes of 22 (suramin) and 23 (nicotinamide) (Figure 12), a focused library of fragments that contain structural features from either suramin or nicotinamide was recently constructed and biochemically evaluated, leading to the discovery of compound 25 with potent SIRT2-inhibitory activity (48 nM) and selectivity for SIRT2 over SIRT1 and SIRT3. Compound 25 concentration- and time-dependently increased the in vitro acetylation level of α-tubulin (a wellvalidated SIRT2 substrate). Further kinetic investigation indicated that this molecule functions as a competitive binder with respect to peptide substrate and most likely as a noncompetitive binder with respect to NAD+.32 These molecules represent leads for an important new class of

Figure 9. Selective aldo-keto reductase AKR1C1 and AKR1C3 inhibitor.

To find novel butyrylcholinesterase (BChE) inhibitors, a hierarchical virtual screening protocol was performed followed by bioactivity assay of an initial subset of top-ranked molecules (40 members). Compound (+)-14 was found to be the most active, inhibiting BChE at low nanomolar concentration (IC50 = 21.3 nM), inhibiting amyloid β1−42 peptide self-induced aggregation into fibrils by 61.7% at 10 μM and protecting SHSY5Y cells against amyloid-β-induced toxicity. The structure of human BChE complexed with (+)-14 (dissociation constant of 2.7 nM) was solved (Figure 10) and is expected to be useful for optimization of small-molecular BChE inhibitors.26 It should be noted that molecular docking may not reflect the real physical process of binding and in some cases may generate questionable structures despite a high score.27 Issues such as conformational flexibility of protein and conformational change upon ligand binding (induced fit) should be considered in docking simulation, and consequently computational algorithms can be extremely resource-intensive.27 One of the key limitations in target-based de novo drug design is the limited number of available crystal structures of F



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Figure 11. Substrate-based approach to design of isoform-selective inhibitors of epigenetic enzymes. The initial substrates 15 and 25 are used in the commercially available assay kits.

epigenetic modulators with potential application as anticancer agents.

The ability of JHDMs to selectively recognize their targets has been elegantly addressed by means of structural biology G



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Figure 12. Crystal structures of (a) human sirtuin homologue 5 complexed with 26 (PDB code: 2NYR) and (b) sirtuin 2-deacetylated peptide complexed with 23 and 2′-O-acetyl adenosine-5-diphosphoribose (PDB code: 2H4J). (c) Superposition of the binding modes of 22 and 23. For the sake of clarity, amino acid residues in the active site are omitted.

studies.33 On the basis of the enzymatic mechanism and the crystal structures of JHDMs complexed with α-ketoglutarate substrate, a potential JHDM-selective small-molecule inhibitor 26 was identified which contains a methyllysine analogue (substrate mimic), an α-ketoglutarate mimic (cofactor isostere), and a linker combining these two fragments (Figure 11).34 Its methyl ester prodrug, methylstat (27), selectively inhibits JHDMs in cells and may be a useful functional probe in epigenetics studies. In addition, a substrate-based approach (substrate activity screening, SAS) has been employed to uncover novel nonpeptidic selective inhibitors of striatal-enriched protein tyrosine phosphatase (STEP). Optimization of substrate scaffolds (exemplified by 28) led to the discovery of several STEP inhibitors active at single-digit micromolar concentration (exemplified by 29) and with greater than 20-fold selectivity over dual specificity phosphatases and multiple tyrosine phosphatases (Figure 13). Compound 29 significantly inhibited STEP in rat cortical neurons.35 The potency, small size, and synthetic accessibility of this compound class will encourage medicinal chemists to further explore this series of STEP inhibitors.

Figure 13. Fragment-based approach to the design of substratederived STEP-selective inhibitors.

3.2. Bidentate-Binding (Bisubstrate) Inhibitors Exploiting Noncatalytic Interactions. Protein kinases are one of the most heavily hit therapeutic target families, but the high structural and sequence homology of kinase isoforms, especially at the conserved ATP binding site, inherently result in polypharmacology and low selectivity. To find selective kinase inhibitors as both pharmacological tools and safer therapeutic candidates, a bidentate-binding (bivalent) strategy H



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Figure 14. Derivation of 31 from a virtual hit molecule 30 and structure of PDE2A complexed with 31 (PDB code: 4HTX).

Figure 15. Discovery of highly potent and selective fused pyrimidine-based MMP-13 inhibitors without any ZBG.

pocket of MMPs displays the highest variability in shape and length, it has been widely used to modulate ligand selectivity.44 On the basis of the three-dimensional structure of the protein target, a series of potent MMP-13 selective inhibitors was optimized to occupy the distinct deep S1′ pocket and adjacent side pocket. Among them, quinazoline-2-carboxamide derivative 33 exhibited potent inhibitory activity (IC50 = 0.0039 nM) and high selectivity (greater than 41000-fold) over other MMPs (MMP-1, -2, -3, -7, -8, -9, -10, -14, and TACE).45 Further structural evolution of the fused pyrimidine scaffold led to thieno[2,3-d]pyrimidine-2-carboxamides as highly active and selective MMP-13 inhibitors, exemplified by 34 (Figure 15).46 X-ray structure analysis of MMP-13 complexed with 33 (the PDB code was not disclosed) confirmed that this molecule is buried deep in the S1′ pocket owing to a β-sheet-type Hbonding interaction with the main chain of MMP-13 (Thr245 and Thr247) spanning the S1′ pocket and also interacts with an additional S1′ side pocket (S1″ pocket) in MMP-13 without interacting with the catalytic zinc (it does not contain a zinc binding group, ZBG) (Figure 15).45 Furthermore, no overt toxicity was seen in a preliminary repeated-dose oral toxicity study of compound 33 in rats. Monosodium salt of 33 is an orally bioavailable MMP-13 inhibitor and greatly reduced release of degradation products (CTX-II) from articular cartilage into the joint cavity in an in vivo rat MIA model in a single oral dose study.45 Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that function as key regulatory proteins in

that simultaneously utilizes binding at the ATP hinge site and at a unique pocket outside the ATP cleft has been employed.31,36,37−41 The principal advantage of bisubstrate inhibitors is their ability to bind at plural sites on the kinase surface, resulting in increased affinity and selectivity compared with traditional active site-targeted inhibitors.42 In this section, we illustrate the usefulness of this approach by discussing its application to various targets, including kinase. An X-ray crystal structure analysis of phosphodiesterase 2 (PDE2) complexed with a highly selective inhibitor 31 (BAY60−7550),43 a derivative of hit 30, showed that 31 binds not only to the PDE2 active site by employing the traditional glutamine-switch substrate-binding mode but also to an induced hydrophobic site (namely, the H-pocket) that does not exist in apo PDE2A (PDB code: 4HTZ). Affinity profiling by molecular modeling indicated that binding to the H-pocket makes a major contribution to the overall binding affinity and thereby improves the selectivity of this inhibitor for PDE2 (Figure 14).43 This clearly illustrates the value of induced, noncatalytic binding sites for obtaining high selectivity. HTS for matrix metalloproteinase 13 (MMP-13)-inhibitory activity yielded a moderately active quinazoline-2-carboxamide (32) (IC50 = 12 nM) (Figure 15). The X-ray crystal structure of the complex of 32 with MMP-13 catalytic domain (PDB code: 3WV2) revealed the structural basis for the high selectivity.44,45 In MMP−inhibitor complexes, a hydrogen bond is frequently observed between the ligand and the backbone amide of Leu and Ala residues flanking the catalytic site. Because the S1′ I



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Figure 16. Co-crystal structures of CDK2 with hit 35 (PDB code: 3QQK) and diaminothiazole 36 (PDB code: 3QXP), which is a highly selective inhibitor of CDK2/5.

Figure 17. Structure-based design of highly selective PDE9 inhibitor 38 from lead 37 (the selectivity panel data were not provided in the original paper) (a) and the crystal structure of the complex of the PDE9 catalytic domain with 38 (PDB code: 4GH6); (b) for a surface presentation; (c) for a stick model. The Tyr424 is emphasized by large bold font.

progression of the cell cycle. The hit compound 35 discovered by HTS was a weak inhibitor (IC50 = 15 μM). Structure-based

modification of 35 was then conducted, aiming to utilize the untapped chemical space identified in the structure of the J



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Figure 18. Design of a potent and isoform-selective covalent PI3Kα inhibitor 40 and X-ray structure of the complex of PI3Kα with 40 (PDB code: 3ZIM).

3ZIM) confirmed the validity of the design strategy (Figure 18). Compound 40 also potently (EC50 < 100 nM) and selectively inhibited signaling in PI3Kα-dependent cancer cell lines, resulting in robust antiproliferative activity (GI50 < 100 nM).49b Covalent linkage formation has been employed in various approved drugs.50 In general, this strategy requires that the target protein should contain a reactive residue that is unusual or nonconserved among other isoforms. On the basis of the X-ray structure of PI3Kβ complexed with a pan-PI3K inhibitor (PDB: 2Y3A) and the fact that Asp862 is a nonconserved amino acid residue unique to the β isoform (Gln, Lys, and Asn residues occupy this position in PI3Kα, γ, and δ, respectively), a series of aminoacyl-triazines (exemplified by 42) derived from the pan-PI3K inhibitor 41 (ZSTK474) were identified as potent and PI3Kβ-selective inhibitors;51 they also showed strong inhibition of cellular Akt phosphorylation and growth of PTEN-deficient MD-MBA-468 cells (Figure 19).

CDK2 complex with 35 (PDB: 3QQK). This yielded a series of highly potent and selective diaminothiazole inhibitors of CDK2 (IC50 = 0.9−1.5 nM). Notably, compound 36 displayed high selectivity for CDK2 and CDK5 over CDK1, CDK4, CDK6, and CDK9 (Figure 16). Compound 36 suppressed the proliferation of 13 cancer cell lines with IC50 values ranging from 0.27 to 6.9 μM, in line with observations of complete inhibition of retinoblastoma phosphorylation and induction of apoptosis.47 Structure determination of the complex of CDK2 with 36 (PDB code: 3QXP) provided new structural insights into the CDK2 inhibitor selectivity and confirmed the usefulness of structure-based design to obtain isozyme-selective inhibitors. 3.3. Structure-Guided Optimization of Isoform-Preferential Interactions. On the basis of the solved crystal structures of target protein−ligand complexes, favorable and unfavorable interactions can now be analyzed by means of computer-aided molecular modeling and dynamics simulation. Favorable interactions or critical amino acids can also be targeted by designing molecular scaffolds with appropriate three-dimensional shapes to afford increased binding affinity and selectivity. For example, crystallographic analysis of phosphodiesterase-9 (PDE9) identified Tyr424, a unique residue of PDE8 and PDE9, as a potential target for improvement of the selectivity of PDE9 inhibitors. Indeed, on the basis of a combination of structure-based design and computational docking, a new series of PDE9 inhibitors with a 6-amino-pyrazolopyrimidinone scaffold (exemplified by compound 37) was developed. Among them, 38 showed IC50 values of 21 nM and 3.3 μM for PDE9 and PDE5, respectively, with about 3 orders of magnitude selectivity over other PDE isoforms. The structure of the complex of PDE9 catalytic domain with 38 (PDB code: 4GH6) was solved, and it revealed direct H-bonding between Tyr424 and 38, which was considered to account for the high selectivity for PDE9 over other PDE isoforms (Figure 17).48 Isoform-selective antagonists of phosphoinositide 3-kinases (PI3Ks) have potential value in cell biology and clinical practice. For example, PI3Kα is an oncogene in human tumors. A cysteine residue is present in PI3Kα (Cys862) but not in other P13K isoforms. Targeting this cysteine might therefore be a means to achieve selective covalent inhibition of PI3Kα. Through structure-based drug design from 39 (GDC-0941),49a a targeted covalent inhibitor 40 (CNX-1351) was designed and synthesized, and indeed, it potently and selectively inhibited PI3Kα. The results of MS and X-ray crystallography (PDB:

Figure 19. Design of isoform-selective PI3Kβ inhibitor 42 targeting nonconserved Asp862 of PI3Kβ.

Within this series, the L-amino acyl compounds preferentially inhibited PI3Kβ while their D-counterparts preferentially inhibited PI3Kδ. The mechanism of the inhibition was investigated by using site-directed mutants, and Asp862 was confirmed to play a key role. An overlay of the structures of the sulfonylpiperazine skeleton (43) and an HTS hit (44) in their complexes with PI3Kγ displayed a high degree of overlap. This was exploited to design novel hybrid analogues, such as 45, as selective, potent, and orally bioavailable leads (Figure 20).52 K



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Figure 20. (a) Design of novel hybrid molecule 45 as a PI3Kγ-selective inhibitor. (b) Superimposed complexes of PI3Kγ with 43 (pink, PDB code: 4ANV) and with 44 (green, PDB code: 4ANU). (c) Structure of 45 complexed with PI3Kγ (yellow, PDB code: 4ANW). (d) Superposition of all three X-ray crystal structures.

Vps34 (a lipid PI3K class III isoform) is involved in vesicle trafficking and autophagy and is therefore an interesting target for cancer treatment.53 Starting with tetrahydropyrimidopyrimidinone-based hit molecule 46, structural optimization resulted in the identification of Vps34 inhibitors 47 (SAR405) and 48 (Figure 21). These molecules displayed potent inhibition of Vps34 with extremely high selectivity. The X-ray structures of the complexes of these two molecules with human Vps34, in combination with sequence alignment of the catalytic sites of Vps34 and class I PI3K α, β, δ, and γ, demonstrated unique binding modes and molecular interactions of 47 and 48 in the ATP-binding cleft of Vps34, and the results illustrate well how the unique molecular characteristics of the morpholine synthon bestowed selectivity toward class I PI3Ks.54,55 Compound 48 showed promising in vivo PK parameters in mouse and induced sustained inhibition of Vps34 upon acute administration.55 Most of the heat shock protein 90 (HSP90) inhibitors are pan-inhibitors of HSP90α/β and also to some extent the other two HSP90 isoforms, TRAP1 (a mitochondrial HSP90 isoform) and GRP94 (an ER-resident Hsp90 isoform). The recently determined X-ray structure of GRP94 demonstrated

that this protein contains a unique secondary binding site (πrich pocket) that could provide an opportunity to design isoform-selective inhibitors. It was hypothesized that the panHSP90 inhibitor 49 (Radamide, RDA) (Figure 22) analogues containing a more hydrophobic mimic of the quinone connected to resorcinol via a cis-amide bioisostere could afford GRP94-selective inhibitors. Consequently, structure-based modification of 49 (PDB code: 2FXS) was carried out, and compound 50 was obtained as a potent inhibitor of GRP94 with selectivity for GRP94 over HSP90α/β (Figure 22).56 Compound 50 contains a phenyl ring appended to an imidazole ring, which serves as a cis-amide surrogate. The preferred orientation of the phenyl ring was postulated to interact with the unique GRP94 π-rich pocket to achieve excellent selectivity. Compound 50 also inhibited trafficking of Toll-like receptors (TLRs) to the cell membrane of HEK293 cells expressing GRP94 in a cellular assay (IC50 = 32 nM). It did not inhibit cytosolic HSP90α/β client proteins (AKT or CRAF) and had no effect on cell viability in the same concentration range. Through a systematic investigation of the kinome, Pro455 and Asn457 in spleen tyrosine kinase (Syk) ATP binding site were identified as a rare combination among sequence-aligned L



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Figure 21. Design of tetrahydropyrimidopyrimidinone-based selective Vps34 inhibitors and binding modes of compounds 47 and 48 complexed with VPS34 (PDB codes 4OYS and 4UWL, respectively).

kinases. It was hypothesized that the spatial proximity of Pro455 and Asn457 could be utilized to design Syk-selective inhibitors that would interact with both residues simultaneously, affording high selectivity for Syk over other isozymes. On the basis of the above analysis and starting with lead compound 51, thiazolopyrimidine analogue 52 was discovered as a selective Syk inhibitor. The X-ray structure of the complex of 52 (depicted in green) with Syk confirmed that this molecule targeted Pro455 and Asn457 (Figure 23).57 In the overlay of the X-ray structures of the complexes of tyrosine-protein kinase 2 (TYK2) and Janus kinase 2 (JAK2) with the lead molecule 4-aminopyridine benzamide 53 (Figure 24), there is a clear difference of P-loop conformations between TYK2 and JAK2. Further, the positively charged arginine (Arg901) in TYK2 is replaced by neutral glutamine (Gln853)

Figure 22. Design of a selective Grp94 inhibitor 50 targeting the unique π-rich pocket.

Figure 23. Design of thiazolopyrimidine 52. The structure of the complex with Syk (PDB code: 4FYO) is shown with Pro455 and Asn457 highlighted in red. M



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Figure 24. Design of TYK2 selective inhibitor 54 based on structural overlay of TYK2 (PDB code: 4GIH) and JAK2 (PDB code: 4GMY) complexes with lead compound 53 (TYK2 pink, JAK2 green), highlighting the discrepancy of P-loop conformations between TYK2 and JAK2 and the sequence difference (Arg901 versus Gln853) in the cyclopropylamide target site.

Figure 25. (a) X-ray structure of complex of 55 with JAK1 (blue, Glu966 from structure 4EHZ) overlaid with the corresponding structure of JAK2 (yellow, Asp939 from PDB code 4F09) to show the pivotal residue difference. (b) Design of imidazopyrrolopyridine (56) as a JAK1-selective inhibitor. (c) X-ray structure of the complex of 56 with JAK1 (PDB code: 4IVB). (d) X-ray structure of the complex of 56 with JAK2 (PDB code: 4IVA).

in JAK2. In theory, this difference can be used to improve the TYK2 selectivity of 53. Initial modeling showed that substitution at the C2-site of the cyclopropylamide moiety could afford a vector to access this domain. Further structurebased optimization eventually led to the discovery of a highly potent, selective, and orally bioavailable TYK2 inhibitor 54. Compound 54 showed statistically significant knockdown of interferon-γ (IFNγ) in a mouse IL-12 PK/PD model assay, indicating that selective suppression of TYK2 kinase activity should be sufficient to suppress the in vivo IL-12 pathway. This study suggested that selectivity could be achieved not only by

directly targeting sequence differences but also by utilizing the overall discrepancy in P-loop conformation or flexibility.58 X-ray structures of a C-2 methyl analogue 55 complexed with JAK1 (PDB code: 4EHZ) and JAK2 (PDB code: 4F09) revealed differential protein−ligand interactions between these two isozymes, providing an opportunity for improving selectivity (Figure 23). Structure-based design culminated in the identification of a C-2 hydroxyethyl imidazopyrrolopyridine derivative 56 as a potent JAK1 inhibitor with high selectivity over JAK2. Compound 56 showed favorable physicochemical properties in a range of preclinical studies and was active in a N



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Figure 26. Design of JAK3-selective inhibitor 59 via structure-based modification of initial phenyl ether.

Figure 27. Discovery of selective epigenetic inhibitors via structure-based drug design combined with homology modeling.

than with serine (and its water network) in the other JAK isoforms, which might result in improved selectivity. Computational and crystallographic analysis suggested that the phenyl ether moiety of 57 possessed a favorable vector to reach Cys909 (Figure 26). Further computer-aided, structure-based design in the light of these theoretical results led to the discovery of 59, which displayed potent and selective inhibition of JAK3.60 Selective inhibitors of epigenetic proteins are potential therapeutic agents for a diverse range of pathological conditions, including many kinds of cancer. As in the case of the kinase family, all the isoforms of epigenetic proteins usually

rat CIA model. X-ray structures of the complexes of 56 with JAK1 and JAK2 (Figure 25c,d) demonstrated that the C-2 (R)hydroxyethyl moiety interacts differently with the Glu966 and Asp939 residues, accounting for the remarkable selectivity.59 Compounds 57 and 58, two initial leads with the 5Hpyrrolo[2,3-b]pyrazine scaffold, were potent ATP-competitive inhibitors of JAKs but lacked selectivity among JAK isoforms. It was recognized that a cysteine residue (Cys909) in JAK3 is replaced by serine at the same position in both JAK1 and JAK2. On the basis of this difference, it was hypothesized that a hydrophobic moiety located in this area of the binding pocket should form fewer unfavorable contacts with cysteine in JAK3 O



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Figure 28. Design of highly selective salicylketoxime-based estrogen receptor β agonists.

Figure 29. Conformationally restricted bioactive molecules.

bearing a tertiary amine showed 500-fold higher JMJD2Cinhibitory potency and over 9100-fold greater selectivity for JMJD2C than the lead structure NOG (62).62 Similarly, in 2012, in the same lab, homology models of human sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) were used to lead 64 to 3′phenethyloxy-2-anilinobenzamides such as 65 and 66, which exhibit more than 3.5-fold greater SIRT2-inhibitory potency and over 35-fold higher selectivity for SIRT2 compared with 67 (AGK2), a previously identified SIRT2-selective inhibitor.63 The ligand−enzyme interactions were studied in detail by molecular docking to understand the SAR and to obtain structural clues for further optimization. Because differences in the ligand-binding sites of estrogen receptor (ER) subtypes α and β are small, the discovery of subtype-selective inhibitors is particularly challenging. To search for additional preferred interactions that might improve ER binding affinity or selectivity, modeling analysis of the ERβbinding cavity with compound 68 (the simplest member of the salaldox B class of previously developed salicylaldoxime-based ERβ agonists) was performed. The modeling study identified

have nearly identical catalytic domains (active sites), and chemical inhibitors of these epigenetic enzymes usually carry analogous structural motifs, making structure-based isoformselective drug design a challenging problem. In the following section, we examine several scenarios in which crystallography and/or molecular modeling could contribute to the development of selective agents (Figure 27). It was demonstrated by molecular modeling that structural differences in the catalytic channels and rim region of HDAC6 and HDAC1 afforded a potential basis for rational design of isoform-selective HDAC inhibitors. According to the pharmacophore model for the existing HDAC inhibitors (exemplified by SAHA), a series of carbazole hydroxamic acids with alkylaryl and alkyl linkers was prepared, culminating in the discovery of 61 (tubastatin A) as a potent and selective inhibitor of HDAC6 (Figure 27).61 In 2010, rational drug design principles combined with homology modeling of JMJD2 were utilized to develop selective JMJD2 inhibitors based on the structure of Noxalylglycine (NOG, 62). A novel hydroxamate analogue 63 P



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Figure 30. Schematic representation of conformationally restricted aminoglycosides.

respectively) and displayed more than 40-fold selectivity for CBP over the other BRD isoforms.67 The ring-constrained bistriazole compound 73 selectively inhibited plasmin and plasma kallikrein with Ki values of 0.77 and 2.4 nM, respectively, while it displayed low potency against the related trypsin-like serine proteases thrombin, activated protein C, and factor Xa.68 Consideration of the biochemical mechanisms of bacterial resistance indicated that it might be feasible to design a conformationally constrained oligosaccharide that would retain antibiotic activity without being susceptible to enzymatic inactivation (the major biochemical mechanism of bacterial resistance). In the light of this theoretical analysis, several aminoglycosides, including 75 and 76, fixed in the ribosomebound “bioactive” conformation, were prepared based on the lead compound 74 (Figure 30). The conformational constraint had only a modest influence on their binding with rRNA. However, it had a substantial influence on enzymatic inactivation. Thus, rational design of ring-restricted aminoglycosides has been considered to be a novel approach to overcome bacterial resistance.69 Thus, forced adoption of a preferential conformation by appropriate use of chiral centers and macrocyclization can result in higher selectivity. 3.5. Rapid Assembly and Screening of Focused Combinatorial Fragment Libraries. As described above, fragment-based drug discovery is a powerful method to discover small-molecular modulators of biological targets. In recent years, diversity-oriented synthesis (DOS) of small chemical entities based on privileged fragments has emerged as a new strategy for exploring the interactions between chemistry and biology.70 DOS affords spatially well-defined compounds that can be utilized not only as chemical tools for protein targets of interest but also as selective modulators of certain disease processes. Integrated high-throughput synthesis and screening of a focused library based on privileged heterocyclic or peptide fragments has been employed to seek bioactive molecules targeting epigenetic proteins such as the first cyclic tetrapeptide-derived HDAC6-selective inhibitors.71,72 Consequently, it is a key challenge in contemporary drug discovery to develop HTS-amenable organic reactions suitable for high-speed synthesis of diverse chemical libraries. In recent years, new synthetic concepts and methodologies have been developed. Notably, click chemistry is a powerful tool for chemical synthesis with outstanding bioorthogonality properties: it is rapid, biocompatible, and highly specific. Among the extant click reactions, copper(I)-catalyzed azide−alkyne [3 + 2]

an empty hydrophobic subpocket delimited by Leu301 and Phe356 in ERβ around the aldoxime hydrogen atom of compound 68 (Figure 28). Therefore, a suitable small lipophilic group (methyl, ethyl, or trifluoromethyl) was introduced into compound 68 to fill this cavity, culminating in the identification of a new salicylketoxime compound 69a with remarkably high subtype-selective binding affinities for ERβ in both in vitro cellfree assays and cell-based assays.64 In summary, refinement of the basic skeleton of existing bioactive molecules combined with introduction of isoformpreferred substituents is an accepted approach in medicinal chemistry to find novel compounds with increased selectivity. The availability of crystal structures is also useful to provide insights for design of inhibitors with improved selectivity profiles. A key requirement for future work will be to understand in detail how the subtle structural differences between isozymes influence the interaction with ligands. 3.4. Conformational Restriction. Conformation restriction has been widely utilized in contemporary drug design to obtain potent and selective agents. Flexible binders are considered to suffer an entropic penalty upon binding due to the freezing of rotatable bonds. Thus, the conformation restriction strategy may increase inhibitory potency by stabilizing a favorable binding conformation (therefore reducing the entropic penalty on binding to the target) or may improve isoform selectivity by eliminating bioactive conformers that give undesired biological responses. Besides, from the viewpoint of ligand efficiency, conformational control could be a very atom-efficient way to improve binding affinity without introducing additional interactions. Because of these advantages, the conformation restriction strategy has attracted considerable attention in recent drug discovery.65 Compound 70 containing a chiral trans-cyclopropane unit is a selective inhibitor of γ-aminobutyric acid (GABA) transporter (GAT) subtypes BGT-1 (betaine/GABA transporter-1) and GAT-3. Compound 70 was developed by applying a threedimensional, diversity-oriented conformational restriction strategy. Further conformational restriction of 70 with the rigid bicyclo[3.1.0]hexane platform afforded the first highly selective BGT-1 inhibitor 71 (IC50 = 0.59 μM) (Figure 29).66 The structure-based conformational restriction strategy has also been applied to the discovery of highly selective inhibitors of CBP/p300 bromodomains67 and plasmin inhibitors.68 Compound 72, identified by means of a stereochemical (asymmetric geometry) diversity-oriented conformational restriction strategy, was found to be a highly active inhibitor of CBP and p300 BRDs (Kd = 0.021 and 0.032 μM, Q



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Figure 31. Identification of highly selective and potent epigenetic inhibitors by using click chemistry-based combinatorial fragment assembly.

Figure 32. Application of the CuAAC reaction to construct a library of 83-derived triazole derivatives, coupled with rapid screening.

dipolar cycloaddition (CuAAC) is one of the most versatile reactions, allowing for the preparation of 1,4-functionalized triazole products in high yields under mild reaction conditions; it has become one of the most widely used reactions in chemical biology and medicinal chemistry.73 The use of pharmacophores of existing pan-inhibitors is a crucial strategy in generating candidate libraries. From a medicinal chemistry point of view, most HDAC inhibitors fall into a widely accepted pharmacophoric model (Figure 31), which consists of a capping moiety (Cap) capable of interacting with residues on the rim of the HDAC catalytic tunnel, a zincbinding group (ZBG) capable of chelating the Zn2+ ion at the bottom of the catalytic subpocket, and a hydrophobic linker connecting the two (exemplified by SAHA) (Figure 31).2,74 On the basis of this pharmacophoric model, isoform-selective HDAC inhibitors have been identified. Two 1,2,3-triazole-

based libraries of HDAC inhibitor candidates were constructed by using CuAAC reactions between alkynes bearing ZBG as pharmacomodulating elements (warheads) and diversified azide fragments as cap structures. The “clicked” triazole libraries were sufficiently pure and therefore could be used directly for biological screenings (using DMSO stock solutions of compounds), and this led to the identification of HDAC8selective inhibitor 77 (C149) and HDAC3-selective inhibitors 78 (T247) and 79 (T326) (Figure 31).75,76 These compounds also showed selective inhibition of HDAC8 or HDAC3 in cellbased assays and exerted growth-inhibitory effects on cancer cells. Furthermore, computational modeling revealed key interactions between HDAC8/3 and inhibitors, suggesting a rational basis for the observed selectivity. However, it is still particularly challenging to obtain high selectivity among the 11 zinc-dependent HDAC isoforms (for R



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practically attainable libraries. Therefore, rational design of inhibitors targeting isozymes via structure-based or mechanismbased approaches and virtual screening techniques can provide significant benefits. However, this requires a detailed understanding of the interplay among the many factors governing the binding of ligands to the target site. Compared with HTS of drug-like compounds, fragment (substrate) screening has frequently afforded high hit rates against a variety of targets in spite of the smaller size of the libraries. The first issue that needs to be confronted is the specificity of substrates or inhibitor fragments. While the compact structures of fragments can minimize steric hindrance during binding with the target, this may also favor interaction with binding pockets in unrelated targets, resulting in impaired specificity.79 Careful structural analysis of complexes of the target with loose binders could offer clues to a suitable decoration pattern for previously identified substrates or fragments. There are also indications that spatially well-defined compounds (such as the chiral cyclopropane ring) with multiple chiral centers in the substrate or fragment scaffolds may have greater potential for elaboration into selective binders of biotargets.65 Selection of appropriate design principles, such as the bisubstrate (bivalent) approach or conformational restriction, depending upon the nature of the isozyme family in question, is also important in the optimization of existing pan-inhibitors or in the conversion of substrates to selective inhibitors. Exploitation of isoform-preferred interactions also provides a rational tool for structure-guided design of inhibitors with distinct selectivity profiles, especially for proteins with highly similar structures and sequences. These approaches are at the forefront of medicinal chemistry research. A relatively new strategy in the identification of targetspecific or isoform-selective inhibitors of a number of biological targets is the rapid assembly and in situ screening of focused combinatorial fragment libraries. Because a purification procedure is not required after library assembly by means of the CuAAC reaction, medicinal chemists can explore the relevant chemical space around a certain scaffold in a quick and efficient manner. Nevertheless, the possibility of false positives and negatives must be considered. For instance, copper will be present in the samples to be assayed in in situ screening, so it is important to examine the effects of copper salts at the highest expected concentration as a control. Also, it is still necessary to extend the scope of synthetic strategies to generate diverse libraries of alkyne/azide-bearing building blocks (key components in click chemistry). It should be pointed out that optimization of target selectivity should be coupled with optimization of potency. Some of the techniques discussed here might be useful in generating compounds that are both potent and selective. The residence time of a drug on its target after binding is one of the key parameters of sustained efficiency and selectivity. Therefore, the binding kinetics of ligand−target complexes, namely the association rate (kon) and dissociation rate (koff), should be measured at an early stage of drug development. Many approved drugs appear to fall into one of two categories: (i) slow binding but with a long residence time, and (ii) fast binding but with a short residence time.27 In recent years, there has been a growing interest in the discovery of time-dependent selective inhibitors.80,81 Covalent interaction with the target protein offers significant pharmacological advantages. Covalent ligand−target interac-

instance, selectivity between HDAC1 and 2, which show very close structural similarities). Analogously, a series of 114 SIRT inhibitor candidates was prepared using the CuAAC reaction of two 2-anilinobenzamide-containing alkynes (as pharmacophoric elements) with 57 azide building blocks. Two SIRT2-selective binders, 81 (A1B11) and 82 (A2B57), were identified by rapid screening. These compounds were more SIRT2-selective than the existing SIRT2 inhibitor 67 (AGK2).77 These studies in Suzuki’s lab confirmed the robustness and efficiency of the click chemistry approach for lead discovery and structural optimization of isozyme-selective inhibitors targeting epigenetic proteins. As described above, one approach for enhancing the potency and selectivity of initial hit molecules is formation of a covalent bond with a nucleophilic residue that is specific to a desired target and ideally without off-targets. JAK3 is the only Janus kinase containing a cysteine residue in the solvent-exposed front domain. Therefore, introduction of an electrophilic covalent tag to replace the nitrile group in 83 (Ruxolitinib) was considered a promising approach to selectively address this JAK3 isozyme. To prepare a JAK3 screening library of Ruxolitinib-derived molecules lacking the nitrile moiety, the pyrazole core in 83 was replaced with a 1,4-substituted triazole surrogate accessible by means of CuAAC reaction (Figure 32). In parallel syntheses, the most convenient protocol is to perform the reaction in centrifuge tubes, which allowed retrieval of the product by centrifugation. This avoids the need to transfer the suspension after precipitation. Moreover, the centrifugation method avoids the possibility of filter plugging and is very suitable for automation. Application of this approach led to the discovery of an active JAK3 inhibitor 84 (IC50 = 35 nM) with high selectivity over other JAKs isoforms, probably via a covalent binding mechanism.78

4. CONCLUSIONS AND PROSPECTS The generation of potent and selective modulators will continue to be central to deciphering the physiological functions of isozymes and to the development of new therapeutic agents for many diseases. In this Perspective, we have focused on strategies for the discovery of target-specific or isoform-selective inhibitors and have illustrated these strategies by describing interesting examples ranging from structure/ mechanism-based de novo design to rapid assembly and screening of focused combinatorial fragment libraries for the design of potentially highly selective modulators. Further development of these and other strategies or technologies may ultimately allow us to design completely isoform-selective compounds in a rational and effective manner, which has important implications for the discovery of selective modulators of ion channels, nuclear receptors, GPCRs, and many other biotargets. HTS of compound libraries is time- and resource-consuming, and considerable efforts must be invested in trial-and-error screening, profiling, and selecting the most promising candidates for further investigation. On the other hand, dynamic combinatorial chemistry (DCC) employs reversible covalent or noncovalent interactions between a set of small fragments to provide a mixture of reversibly reacting building blocks under thermodynamic control, but, in such a solutionphase approach it is important avoid the use of additional reagents in order to avoid possible adverse effects in subsequent in situ biochemical evaluations, and this in turn limits the choice of applicable reactions, as well as the scale of the S



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tions have the benefit of higher binding energies, prolonged duration of the pharmacological effect (drug-target residence time) and potential for improved selectivity. However, a challenge in developing covalent inhibitors is to identify suitable reactive functional moieties (“warheads”); usually such moieties require a level of electrophilicity that does not make them so reactive as to be promiscuous.82 It should be emphasized that different protein isoforms, e.g., among kinases, HDACs and other epigenetic protein families, exhibit a great variety of tissue distributions, expression profiles, and subcellular locations in healthy versus diseased cells. Usually, these isozymes are components of sophisticated multiprotein complexes in cells, and the observed selectivity of binders or substrates toward isolated enzymes may be different from that toward biomacromolecular complexes in cells, adding a further layer of complexity to the discovery of genuine selective inhibitors.2,83,84 Therefore, screening for inhibitor specificity using multiprotein complexes rather than purified proteins or the addition of requisite protein−protein interaction assays is advocated. Basically, a very detailed understanding of the complex physiology of isoforms, cellular, and cell-type specific protein complexes (protein−protein interactions) and their substrates and modulators at the molecular level plays a central role in the design of highly potent and selective inhibitors. Nevertheless, we believe these problems are not intractable; development of integrated strategies to design highly selective bioactive molecules for specific molecular recognition in complex biological systems is already under way and may ultimately make it feasible to precisely manipulate and control many biological processes.

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bioactive small molecules, analysis of the mechanisms of their interaction with biological targets, and mechanisms of pharmacological action. Takayoshi Suzuki received his B.Sc. and M.Sc. from the University of Tokyo (1995 and 1997, respectively) and then became a Researcher at Japan Tobacco Inc. (1997−2002). He subsequently joined the Graduate School of Pharmaceutical Sciences, Nagoya City University, as an Assistant Professor (2003−2009) and Lecturer (2009−2011). During that time, he received his Ph.D. from the Graduate School of Pharmaceutical Sciences, the University of Tokyo. He is currently a full Professor at the Graduate School of Medical Science, Kyoto Prefectural University of Medicine (2011 to present). His research interests are in the areas of medicinal chemistry and bioorganic chemistry. Xinyong Liu received his B.Sc. and M.Sc. degrees from School of Pharmaceutical Sciences, Shandong University, in 1984 and in 1991, respectively. From 1997 to 1999 he worked at Instituto de Quimica Medica (CSIC) in Spain as a senior visiting scholar. He obtained his Ph.D. from Shandong University in 2004. He is currently a full Professor of the Institute of Medicinal Chemistry, Shandong University. His research interests include rational drug design, synthesis, and antiviral evaluation of a variety of molecules that interact with specific enzymes and receptors in the viral life cycle (HIV, HBV, HCV, and FluV). Other ongoing programs include the molecular modification and structure−activity relationships study of some natural products to treat cardiovascular diseases and drug delivery research using PEGylated small-molecular agents.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (NSFC nos. 81102320, 81273354), Key Project of NSFC for International Cooperation (no. 81420108027), Research Fund for the Doctoral Program of Higher Education of China (nos. 20110131130005, 20110131120037), The Science and Technology Development Project of Shandong Province (no. 2014GSF118012), China Postdoctoral Science Foundation funded special project (no. 2012T50584), Postdoctoral Fellow Allowance (ID no. P12418) from Japan Society for the Promotion of Science (JSPS), JST CREST program, Takeda Science Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Scientific Research from JSPS, the Tokyo Biochemical Research Foundation (T.S.), and the Uehara Memorial Foundation (T.S.).

AUTHOR INFORMATION

Corresponding Authors

*For P.Z.: E-mail, [email protected]. *For T.Z.: E-mail, [email protected]. *For X.-Y.L.: E-mail, [email protected]. Notes

The authors declare no competing financial interest. All figures showing binding modes were produced with PyMol (www.pymol.org). Biographies Peng Zhan obtained his B.Sc. degree from Shandong University, China, in 2005. Then he earned his M.Sc. degree and Ph.D. in medicinal chemistry from Shandong University in 2008 and 2010, respectively. He subsequently joined the research group of Professor Xinyong Liu as a Lecturer (2010−2012). From 2012 to 2014, he worked as a Postdoctoral Fellow funded by JSPS (Japan Society for the Promotion of Science) in the Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan. He is now working as an associate professor in the lab of Professor Xinyong Liu. His research interests involve the discovery of novel antiviral, anticancer, and neurodegenerative diseases-related agents based on rational drug design and combinatorial chemistry approaches.

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ABBREVIATIONS USED AChBPs, acetylcholine-binding protein templates; BChE, butyrylcholinesterase; CA, carbonic anhydrase; CDKs, cyclindependent kinases; CuAAC, copper(I) catalyzed azide−alkyne [3 + 2] dipolar cycloaddition; DCC, dynamic combinatorial/ covalent chemistry; DCLs, dynamic constitutional libraries; DOS, diversity-oriented synthesis; ER, estrogen receptor; FBDD, fragment-based drug discovery; GABA, γ-aminobutyric acid; GAT, GABA transporter; HDACs, histone deacetylases; HSP90, heat shock protein 90; HTS, high-throughput screening; IFNγ, interferon-γ; JAK2, Janus kinase 2; JHDMs, Jumonji C domain-containing histone demethylases; LSD1, lysinespecific demethylase 1; MAOs, monoamine oxidases; MMPs, matrix metalloproteinases; NOG, N-oxalylglycine; NOS, nitric oxide synthase; nNOS, neuronal nitric oxide synthase; PCPA, phenylcyclopropylamine; PDEs, phosphodiesterases; PI3K, phosphoinositide-3 kinase; SIRTs, sirtuins; TGS, target-guided

Yukihiro Itoh obtained his Ph.D. in pharmaceutical sciences from the University of Tokyo (2011). Subsequently, he worked as a research associate at the Scripps Research Institute (2011−2012). During that time, he was a Research Fellow of the Japan Society for the Promotion of Science (2010−2012). He is currently an Assistant Professor at the Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan (2012 to present). His research interests are focused on medicinal chemistry and chemical biology, including synthesis of T



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Miyata, N.; Suzuki, T. Lysine-specific demethylase 1-selective inactivators: protein-targeted drug delivery mechanism. Angew. Chem., Int. Ed. Engl. 2013, 52, 8620−8624. (17) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radić, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem., Int. Ed. Engl. 2002, 41, 1053−1057. (18) Manetsch, R.; Krasiński, A.; Radić, Z.; Raushel, J.; Taylor, P.; Sharpless, K. B.; Kolb, H. C. In situ click chemistry: enzyme inhibitors made to their own specifications. J. Am. Chem. Soc. 2004, 126, 12809− 12818. (19) Nasr, G.; Petit, E.; Vullo, D.; Winum, J. Y.; Supuran, C. T.; Barboiu, M. Carbonic anhydrase-encoded dynamic constitutional libraries: toward the discovery of isozyme-specific inhibitors. J. Med. Chem. 2009, 52, 4853−4859. (20) Yamauchi, J. G.; Gomez, K.; Grimster, N.; Dufouil, M.; Nemecz, A.; Fotsing, J. R.; Ho, K. Y.; Talley, T. T.; Sharpless, K. B.; Fokin, V. V.; Taylor, P. Synthesis of selective agonists for the α7 nicotinic acetylcholine receptor with in situ click-chemistry on acetylcholinebinding protein templates. Mol. Pharmacol. 2012, 82, 687−699. (21) Suzuki, T.; Ota, Y.; Kasuya, Y.; Mutsuga, M.; Kawamura, Y.; Tsumoto, H.; Nakagawa, H.; Finn, M. G.; Miyata, N. An unexpected example of protein-templated click chemistry. Angew. Chem., Int. Ed. Engl. 2010, 49, 6817−6820. (22) Danishuddin, M.; Khan, A. U. Structure based virtual screening to discover putative drug candidates: necessary considerations and successful case studies. Methods 2015, 71C, 135−145. (23) Braga, R. C.; Alves, V. M.; Silva, A. C.; Nascimento, M. N.; Silva, F. C.; Liao, L. M.; Andrade, C. H. Virtual screening strategies in medicinal chemistry: the state of the art and current challenges. Curr. Top Med. Chem. 2014, 14, 1899−1912. (24) Lionta, E.; Spyrou, G.; Vassilatis, D. K.; Cournia, Z. Structurebased virtual screening for drug discovery: principles, applications and recent advances. Curr. Top Med. Chem. 2014, 14, 1923−1938. (25) Brožič, P.; Turk, S.; Adeniji, A. O.; Konc, J.; Janežič, D.; Penning, T. M.; Lanišnik Rižner, T.; Gobec, S. Selective inhibitors of aldo-keto reductases AKR1C1 and AKR1C3 discovered by virtual screening of a fragment library. J. Med. Chem. 2012, 55, 7417−7424. (26) Brus, B.; Košak, U.; Turk, S.; Pišlar, A.; Coquelle, N.; Kos, J.; Stojan, J.; Colletier, J. P.; Gobec, S. Discovery, biological evaluation, and crystal structure of a novel nanomolar selective butyrylcholinesterase inhibitor. J. Med. Chem. 2014, 57, 8167−8179. (27) Chen, Y. Beware of docking! Trends Pharmacol. Sci. 2015, 36, 78−95. (28) Wang, T.; Wu, M. B.; Chen, Z. J.; Chen, H.; Lin, J. P.; Yang, L. R. Fragment-based drug discovery and molecular docking in drug design. Curr. Pharm. Biotechnol. 2015, 16, 11−25. (29) Suzuki, T.; Kouketsu, A.; Itoh, Y.; Hisakawa, S.; Maeda, S.; Yoshida, M.; Nakagawa, H.; Miyata, N. Highly potent and selective histone deacetylase 6 inhibitors designed based on a small-molecular substrate. J. Med. Chem. 2006, 49, 4809−4812. (30) Itoh, Y.; Suzuki, T.; Kouketsu, A.; Suzuki, N.; Maeda, S.; Yoshida, M.; Nakagawa, H.; Miyata, N. Design, synthesis, structure− selectivity relationship, and effect on human cancer cells of a novel series of histone deacetylase 6-selective inhibitors. J. Med. Chem. 2007, 50, 5425−5438. (31) Asaba, T.; Suzuki, T.; Ueda, R.; Tsumoto, H.; Nakagawa, H.; Miyata, N. Inhibition of human sirtuins by in situ generation of an acetylated lysine−ADP−ribose conjugate. J. Am. Chem. Soc. 2009, 131, 6989−6996. (32) Cui, H.; Kamal, Z.; Ai, T.; Xu, Y.; More, S. S.; Wilson, D. J.; Chen, L. Discovery of potent and selective sirtuin 2 (SIRT2) inhibitors using a fragment-based approach. J. Med. Chem. 2014, 57, 8340−8357. (33) Pilka, E. S.; James, T.; Lisztwan, J. H. Structural definitions of Jumonji family demethylase selectivity. Drug Discovery Today. 2014, 20, 743−749, DOI: 10.1016/j.drudis.2014.12.013. (34) Luo, X.; Liu, Y.; Kubicek, S.; Myllyharju, J.; Tumber, A.; Ng, S.; Che, K. H.; Podoll, J.; Heightman, T. D.; Oppermann, U.; Schreiber, S.

synthetic methodology; TLRs, Toll-like receptors; STEP, striatal-enriched protein tyrosine phosphatase; Syk, spleen tyrosine kinase; TNKSs, tankyrases; TYK2, tyrosine-protein kinase 2; ZBG, zinc-binding group

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