High affinity targets of protein kinase inhibitors have similar residues at the positions energetically important for binding

Inhibition of protein kinase activity is a focus of intense drug discovery efforts in several therapeutic areas. Major challenges facing the field include understanding of the factors determining the selectivity of kinase inhibitors and the development of compounds with the desired selectivity profile. Here, we report the analysis of sequence variability among high and low affinity targets of eight different small molecule kinase inhibitors (BIRB796, Tarceva, NU6102, Gleevec, SB203580, balanol, H89, PP1). It is observed that all high affinity targets of each inhibitor are found among a relatively small number of kinases, which have similar residues at the specific positions important for binding. The findings are highly statistically significant, and allow one to exclude the majority of kinases in a genome from a list of likely targets for an inhibitor. The findings have implications for the design of novel inhibitors with a desired selectivity profile (e.g. targeted at multiple kinases), the discovery of new targets for kinase inhibitor drugs, comparative analysis of different in vivo models, and the design of "a-la-carte" chemical libraries tailored for individual kinases.     

Measuring and interpreting the selectivity of protein kinase inhibitors

Protein kinase inhibitors are a well-established class of clinically useful drugs, particularly for the treatment of cancer. Achieving inhibitor selectivity for particular protein kinases often remains a significant challenge in the development of new small molecules as drugs or as tools for chemical biology research. This review summarises the methodologies available for measuring kinase inhibitor selectivity, both in vitro and in cells. The interpretation of kinase inhibitor selectivity data is discussed, particularly with reference to the structural biology of the protein targets. Measurement and prediction of kinase inhibitor selectivity will be important for the development of new multi-targeted kinase inhibitors.     

Targeting the Ion Channel Kv1.3 with Scorpion Venom Peptides Engineered for Potency,Selectivity, and Half-life

Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.     

Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.     

Kinomics-structural biology and chemogenomics of kinase inhibitors and targets

Classifying kinases based entirely on small molecule selectivity data is a new approach to drug discovery that allows scientists to understand relationships between targets. This approach combines the understanding of small molecules and targets, and thereby assists the researcher in finding new targets for existing molecules or understanding selectivity and polypharmacology of molecules in related targets. Currently, structural information is available for relatively few of the protein kinases encoded in the human genome (7% of the estimated 518); however, even the current knowledge base, when paired with structure-based design techniques, can assist in the identification and optimization of novel kinase inhibitors across the entire protein class. Chemogenomics attempts to combine genomic data, structural biological data, classical dendrograms, and selectivity data to explore, define, and classify the medicinally relevant kinase space. Exploitation of this information in the discovery of kinase inhibitors defines practical kinase chemogenomics (kinomics). In this paper, we review the available information on kinase targets and their inhibitors, and present the relationships between the various classification schema for kinase space. In particular, we present the first dendrogram of kinases based entirely on small molecule selectivity data. We find that the selectivity dendrogram differs from sequence-based clustering mostly in the higher-level groupings of the smaller clusters, and remains very comparable for closely homologous targets. Highly homologous kinases are, on average, inhibited comparably by small molecules. This observation, although intuitive, is very important to the process of target selection, as one would expect difficulty in achieving inhibitor selectivity for kinases that share high sequence identity.     

Pacifastin-related peptides: structural and functional characteristics of a family of serine peptidase inhibitors

Members of the pacifastin family are serine peptidase inhibitors, found in arthropods and have many members within different insect orders. Based on their structural characteristics, inhibitors of this peptide family are divided into two groups (I and II). Members of both groups exhibit specificity towards different types of serine peptidases. In addition, group I inhibitors display species selectivity. The specificity and selectivity of these inhibitors depends on the nature of their P1 residue and on additional interaction sites at the inhibitor's surface. Functional analysis studies have shown that crustacean pacifastin plays a key role in the immune response, whereas insect pacifastin-like peptides have multiple regulatory functions in processes involved in immunity, reproduction, phase transition, etc.     

Biochemical and pharmacological profiling of the pro-survival protein Bcl-xL

Bcl-2 family proteins are key mediators of programmed cell death. Over-expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) has been associated with tumor progression and chemotherapeutic resistance. Pharmacological agents that neutralize the functions of anti-apoptotic Bcl-2 family proteins have emerged as a promising new class of anti-cancer agents. Biochemical analyses have demonstrated that small molecule inhibitors and some pro-apoptotic proteins exhibit distinct binding preferences for anti-apoptotic proteins. While numerous structures of anti-apoptotic proteins bound to ligands have been reported, the source of this selectivity is still unclear. Here, we present a systematic analysis of a series of Bcl-xL variants that contain mutations within the hydrophobic ligand-binding cleft. The ability of these Bcl-xL mutants to interact with both small molecule inhibitors and BH3 peptides was determined. These studies provide information on the contributions of specific residues to small molecule inhibitor binding and shed light on the ligand selectivity of these therapeutically important proteins.     

The structural requirements of histone deacetylase inhibitors: SAHA analogs modified at the C5 position display dual HDAC6/8 selectivity

Histone deacetylase (HDAC) proteins have emerged as important targets for anti-cancer drugs, with four small molecules approved for use in the clinic. Suberoylanilide hydroxamic acid (Vorinostat, SAHA) was the first FDA-approved HDAC inhibitor for cancer treatment. However, SAHA inhibits most of the eleven HDAC isoforms. To understand the structural requirements of HDAC inhibitor selectivity and develop isoform selective HDAC inhibitors, SAHA analogs modified in the linker at the C5 position were synthesized and tested for potency and selectivity. C5-modified SAHA analogs displayed dual selectivity to HDAC6 and HDAC8 over HDAC 1, 2, and 3, with only a modest reduction in potency. These findings are consistent with prior work showing that modification of the linker region of SAHA can alter isoform selectivity. The observed HDAC6/8 selectivity of C5-modified SAHA analogs provide guidance toward development of isoform selective HDAC inhibitors and more effective anti-cancer drugs.     

Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.     

A Targeted Library Screen Reveals a New Inhibitor Scaffold for Protein Kinase D

Protein kinase D (PKD) has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.     

A profiling platform for the identification of selective metalloprotease inhibitors

Although proteases represent an estimated 5% to 10% of potential drug targets, inhibitors for metalloproteases (MPs) account for only a small proportion of all approved drugs, failures of which have typically been associated with lack of selectivity. In this study, the authors describe a novel and universal binding assay based on an actinonin derivative and show its binding activities for several MPs and its lack of activity toward all the non-MPs tested. This newly developed assay would allow for the rapid screening for inhibitors of a given MP and for the selectivity profiling of the resulting hits. The assay has successfully enabled for the first time simultaneous profiling of 8 well-known inhibitors against a panel of selected MPs. Previously published activities for these inhibitors were confirmed, and the authors have also discovered new molecular targets for some of them. The authors conclude that their profiling platform provides a generic assay solution for the identification of novel metalloprotease inhibitors as well as their selectivity profiling using a simple and homogeneous assay.     

A quantitative analysis of kinase inhibitor selectivity

Kinase inhibitors are a new class of therapeutics with a propensity to inhibit multiple targets. The biological consequences of multi-kinase activity are poorly defined, and an important step toward understanding the relationship between selectivity, efficacy and safety is the exploration of how inhibitors interact with the human kinome. We present interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome. The data constitute the most comprehensive study of kinase inhibitor selectivity to date and reveal a wide diversity of interaction patterns. To enable a global analysis of the results, we introduce the concept of a selectivity score as a general tool to quantify and differentiate the observed interaction patterns. We further investigate the impact of panel size and find that small assay panels do not provide a robust measure of selectivity.     

Crystals of urokinase type plasminogen activator complexes reveal the binding mode of peptidomimetic inhibitors

Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility. In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target. We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.g. phenethylsulfonamidino(P4)-D-seryl(P3)-L-alanyl(P2)-L-argininal(P1) and derivatives thereof) in complex with the uPA proteinase domain. These potent inhibitors are highly selective for uPA. The non-natural D-seryl residue present at the P3 position in these inhibitors contributes substantially to both potency and selectivity because, due to its D-configuration, its side-chain binds in the S4 pocket to interact with the uPA unique residues Leu97b and His99. Additional potency and selectivity can be achieved by optimizing the inhibitor P4 residue to bind a pocket, known as S1sub or S1beta, that is adjacent to the primary specificity pocket of uPA.     

Discovery of 2-amino-heteroaryl-benzothiazole-6-anilides as potent p56(lck) inhibitors

A series of structurally novel benzothiazole based small molecule inhibitors of p56(lck) was prepared to elucidate their structure-activity relationships (SAR), selectivity and cell activity in the T-cell proliferation assay. BMS-350751 (2) and BMS-358233 (3) are identified as potent Lck inhibitors with excellent cellular activities against T-cell proliferation.     

3-Mercaptopropionic acids as efficacious inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa)

A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (-)2a displays TAFIa IC50 = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.     

Discovering potent and selective reversible inhibitors of enzymes in complex proteomes

To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), triacylglycerol hydrolase (TGH) and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.     

Synthesis and inhibitory effect of piperine derivates on monoamine oxidase

A series of piperine derivates (1-19) have been designed, synthesized and evaluated in vitro for their monoamine oxidase (MAO) A and B inhibitory activity and selectivity. It is worth noting that most of the small amine moieties substituted on the piperidine ring proved to be potent and selective inhibitors of MAO-B rather than of MAO-A. 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid n-propyl amide (3) showed the greatest MAO-B inhibitory activity (IC(50)(MAO-B)=0.045 μM) and good selectivity (IC(50)(MAO-A)=3.66 μM). The conjugated double bond and carbonyl group of piperine are proved to be an essential feature for piperine and related alkylamides to exhibit MAO-inhibitory activity. Binding mode of the titled compounds was predicted using FlexX algorithm. The design and optimization of novel small molecule monoamine oxidase inhibitors will be guided by the results of this report.     

Structure-reactivity relationships for the inhibition mechanism at the second alkyl-chain-binding site of cholesterol esterase and lipase.

Alkyl-N-phenyl carbamates (2-8) (see Figure 1), alkyl-N-phenyl thiocarbamates (9-15), 2,2'-biphenyl-2-ol-2'-N-substituted carbamates (16-23), and 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-substituted carbamates (24-31) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase and Pseudomona species lipase. All inhibitors are characterized as transient or pseudo substrate inhibitors for both enzymes. Both enzymes are not protected from inhibition and further inactivated by carbamates 2-8 and thiocarbamates 9-15 in the presence of trifluoroacetophenone. Therefore, carbamates 2-8 and thiocarbamates 9-15 are exceptions for active site binding inhibitors and are probably the second alkyl-chain binding-site-directed inhibitors for both enzymes. The inhibition data for carbamates 2-8 and thiocarbamates 9-15 are correlated with the steric constant, E(s), and the hydrophobicity constant, pi; however, the inhibition data are not correlated with the Taft substituent constant, sigma. A comparison of the inhibition data for carbamates 2-8 and thiocarbamates 9-15 toward both enzymes indicates that thiocarbamates 9-15 are more potent inhibitors than carbamates 2-8. A comparison of the inhibition data for cholesterol esterase and Pseudomona species lipase by carbamates 2-8 or thiocarbamates 9-15 indicates that cholesterol esterase is more sensitive to the E(s) and pi values than Pseudomona species lipase. The negative slope values for the logarithms of inhibition data for Pseudomona species lipase by carbamates 2-8 and thiocarbamates 9-15 versus E(s) and pi indicate that the second alkyl-chain-binding site of Pseudomona species lipase is huge, hydrophilic, compared to that of cholesterol esterase, and prefers to interact with a bulky, hydrophilic inhibitor rather than a small, hydrophobic one. On the contrary, the second alkyl-chain-binding site of cholesterol esterase prefers to bind to a small, hydrophobic inhibitor. Both enzymes are protected from inhibition by carbamates 16-23 in the presence of trifluoroacetophenone. Therefore, carbamates 16-23 are characterized as the alkyl chain binding site, esteratic site oxyanion active site directed pseudo substrate inhibitors for both enzymes. Both enzyme inhibition data for carbamates 16-22 are well-correlated with sigma alone. The negative rho values for these correlations indicate that the serine residue of both enzymes and carbamates 16-22 forms the tetrahedral species with more positive charges than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges than the tetrahedral species. Carbamates 24-31 are also exceptions for active site binding inhibitors and probably the second alkyl chain binding site-directed inhibitors for both enzymes. However, the enzyme inhibition constants for carbamates 24-31 are correlated with values of sigma, E(s), and pi. The negative rho values for these correlations indicate that both enzymes and carbamates 24-31 form the tetrahedral species with more positive charges than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges than those tetrahedral species. Therefore, carbamates 24-31 may bind to both the active sites and the second alkyl chain binding site and follow the evacuation of the active sites. A comparison of the rho values for cholesterol esterase and Pseudomona species lipase by carbamates 24-31 indicates that cholesterol esterase is much more sensitive to the sigma values than Pseudomona species lipase. The negative sensitivity values, delta, for the cholesterol esterase inhibitions by carbamates 24-31 indicate that the enzyme prefers to bind to a bulky carbamyl group rather than bind to a small one. The hydrophobicity of carbamates 24-31 does not play a major role in both enzyme inhibitions.     

Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.     

A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i

Inhibition of the immunoproteasome subunit β5i alleviates autoimmune diseases in preclinical studies and represents a promising new anti‐inflammatory therapy. However, the lack of structural data on the human immunoproteasome still hampers drug design. Here, we systematically determined the potency of seven α' β' epoxyketone inhibitors with varying N‐caps and P3‐stereochemistry for mouse/human β5c/β5i and found pronounced differences in their subunit and species selectivity. Using X‐ray crystallography, the compounds were analyzed for their modes of binding to chimeric yeast proteasomes that incorporate key parts of human β5c, human β5i or mouse β5i and the neighboring β6 subunit. The structural data reveal exceptional conformations for the most selective human β5i inhibitors and highlight subtle structural differences as the major reason for the observed species selectivity. Altogether, the presented results validate the humanized yeast proteasome as a powerful tool for structure‐based development of β5i inhibitors with potential clinical applications.