From Cancer to Pain Target by Automated Selectivity Inversion of a Clinical Candidate.

Elimination of inadvertent binding is crucial for inhibitor design targeting conserved protein classes like kinases. Compounds in clinical trials provide a rich source for initiating drug design efforts by exploiting such secondary binding events. Considering both aspects, we shifted the selectivity of tozasertib, originally developed against AurA as cancer target, toward the pain target TrkA. First, selectivity-determining features in binding pockets were identified by fusing interaction grids of several key and off-target conformations. A focused library was subsequently created and prioritized using a multiobjective selection scheme that filters for selective and highly active compounds based on orthogonal methods grounded in computational chemistry and machine learning. Eighteen high-ranking compounds were synthesized and experimentally tested. The top-ranked compound has 10000-fold improved selectivity versus AurA, nanomolar cellular activity, and is highly selective in a kinase panel. This was achieved in a single round of automated in silico optimization, highlighting the power of recent advances in computer-aided drug design to automate design and selection processes.

KinMap: a web-based tool for interactive navigation through human kinome data.

BACKGROUND: Annotations of the phylogenetic tree of the human kinome is an intuitive way to visualize compound profiling data, structural features of kinases or functional relationships within this important class of proteins. The increasing volume and complexity of kinase-related data underlines the need for a tool that enables complex queries pertaining to kinase disease involvement and potential therapeutic uses of kinase inhibitors. RESULTS: Here, we present KinMap, a user-friendly online tool that facilitates the interactive navigation through kinase knowledge by linking biochemical, structural, and disease association data to the human kinome tree. To this end, preprocessed data from freely-available sources, such as ChEMBL, the Protein Data Bank, and the Center for Therapeutic Target Validation platform are integrated into KinMap and can easily be complemented by proprietary data. The value of KinMap will be exemplarily demonstrated for uncovering new therapeutic indications of known kinase inhibitors and for prioritizing kinases for drug development efforts. CONCLUSION: KinMap represents a new generation of kinome tree viewers which facilitates interactive exploration of the human kinome. KinMap enables generation of high-quality annotated images of the human kinome tree as well as exchange of kinome-related data in scientific communications. Furthermore, KinMap supports multiple input and output formats and recognizes alternative kinase names and links them to a unified naming scheme, which makes it a useful tool across different disciplines and applications. A web-service of KinMap is freely available at http://www.kinhub.org/kinmap/ .

Pocketome of human kinases: prioritizing the ATP binding sites of (yet) untapped protein kinases for drug discovery.

Protein kinases are involved in a variety of diseases including cancer, inflammation, and autoimmune disorders. Although the development of new kinase inhibitors is a major focus in pharmaceutical research, a large number of kinases remained so far unexplored in drug discovery projects. The selection and assessment of targets is an essential but challenging area. Today, a few thousands of experimentally determined kinase structures are available, covering about half of the human kinome. This large structural source allows guiding the target selection via structure-based druggability prediction approaches such as DoGSiteScorer. Here, a thorough analysis of the ATP pockets of the entire human kinome in the DFG-in state is presented in order to prioritize novel kinase structures for drug discovery projects. For this, all human kinase X-ray structures available in the PDB were collected, and homology models were generated for the missing part of the kinome. DoGSiteScorer was used to calculate geometrical and physicochemical properties of the ATP pockets and to predict the potential of each kinase to be druggable. The results indicate that about 75% of the kinome are in principle druggable. Top ranking structures comprise kinases that are primary targets of known approved drugs but additionally point to so far less explored kinases. The presented analysis provides new insights into the druggability of ATP binding pockets of the entire kinome. We anticipate this comprehensive druggability assessment of protein kinases to be helpful for the community to prioritize so far untapped kinases for drug discovery efforts.

Design, synthesis, and evaluation of new thiadiazole-based direct inhibitors of enoyl acyl carrier protein reductase (InhA) for the treatment of tuberculosis.

Mycobacterial enoyl acyl carrier protein reductase (InhA) is a clinically validated target for the treatment of tuberculosis infections, a disease that still causes the death of at least a million people annually. A known class of potent, direct, and competitive InhA inhibitors based on a tetracyclic thiadiazole structure has been shown to have in vivo activity in murine models of tuberculosis infection. On the basis of this template, we have here explored the medicinal chemistry of truncated analogues that have only three aromatic rings. In particular, compounds 8b, 8d, 8f, 8l, and 8n show interesting features, including low nanomolar InhA IC50, submicromolar antimycobacterial potency, and improved physicochemical profiles in comparison with the tetracyclic analogues. From this series, 8d is identified as having the best balance of potency and properties, whereby the resolved 8d S-enatiomer shows encouraging in vivo efficacy.

Discovery, biological evaluation, and crystal structure of a novel nanomolar selective butyrylcholinesterase inhibitor.

Butyrylcholinesterase (BChE) is regarded as a promising drug target as its levels and activity significantly increase in the late stages of Alzheimer's disease. To discover novel BChE inhibitors, we used a hierarchical virtual screening protocol followed by biochemical evaluation of 40 highest scoring hit compounds. Three of the compounds identified showed significant inhibitory activities against BChE. The most potent, compound 1 (IC50 = 21.3 nM), was resynthesized and resolved into its pure enantiomers. A high degree of stereoselective activity was revealed, and a dissociation constant of 2.7 nM was determined for the most potent stereoisomer (+)-1. The crystal structure of human BChE in complex with compound (+)-1 was solved, revealing the binding mode and providing clues for potential optimization. Additionally, compound 1 inhibited amyloid ß(1-42) peptide self-induced aggregation into fibrils (by 61.7% at 10 µM) and protected cultured SH-SY5Y cells against amyloid-ß-induced toxicity. These data suggest that compound 1 represents a promising candidate for hit-to-lead follow-up in the drug-discovery process against Alzheimer's disease.

Endocrine disruptome--an open source prediction tool for assessing endocrine disruption potential through nuclear receptor binding.

Predicting the endocrine disruption potential of compounds is a daunting but essential task. Here we report a new tool for this purpose that we have termed Endocrine Disruptome. It is a free and simple-to-use Web service that runs on an open source platform called Docking interface for Target Systems (DoTS). The molecular docking is handled via AutoDock Vina. Compounds are docked to 18 integrated and well-validated crystal structures of 14 different human nuclear receptors: androgen receptor; estrogen receptors α and ß; glucocorticoid receptor; liver X receptors α and ß; mineralocorticoid receptor; peroxisome proliferator activated receptors α, ß/δ, and γ; progesterone receptor; retinoid X receptor α; and thyroid receptors α and ß. Endocrine Disruptome is free of charge and available at http://endocrinedisruptome.ki.si.

Novel toll-like receptor 4 (TLR4) antagonists identified by structure- and ligand-based virtual screening.

Toll-like receptor 4 (TLR4) in complex with its accessory protein MD-2 represents an emerging target for the treatment of severe sepsis and neuropathic pain. We performed structure-based and ligand-based virtual screening targeting the TLR4-MD-2 interface. Three in silico hit compounds showed promising TLR4 antagonistic activities with micromolar IC50 values. These compounds also suppressed cytokine secretion by human peripheral blood mononuclear cells. The specific affinity of the most potent hit was confirmed by surface plasmon resonance direct-binding experiments. The results of our study represent a very promising starting point for the development of potent small-molecule antagonists of TLR4.

2,3-Diarylpropenoic acids as selective non-steroidal inhibitors of type-5 17ß-hydroxysteroid dehydrogenase (AKR1C3).

The aldo-keto reductase AKR1C3 is an important target for the development of new drugs. Selective inhibitors of this enzyme are needed because they should not inhibit other, structurally closely related AKR1C isoforms. A comprehensive series of 2,3-diarylpropenoic acids was synthesized and evaluated for the inhibition of AKR1C1-AKR1C3. We found that the 4-methylsulfonylphenyl substituent at position 2 of these acids is required to exhibit the selective inhibition of AKR1C3. The best results were obtained for the compounds that fulfill the above requirement and possess a 4-bromophenyl, 4-methylthiophenyl, 4-methylphenyl or 4-ethylphenyl substituent at position 3 of the substituted propenoic acids (i.e., acids 28, 29, 37, and 39, respectively). These compounds represent an important step toward the development of drug candidates for a treatment of the hormone-dependent and hormone-independent forms of prostate and breast cancers.

Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.

Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.

The influence of differential processing of procathepsin H on its aminopeptidase activity, secretion and subcellular localization in human cell lines.

Cathepsin H is a unique member of the cysteine cathepsins that acts primarily as an aminopeptidase. Like other cysteine cathepsins, it is synthesized as an inactive precursor and activated by proteolytic removal of its propeptide. Here we demonstrate that, in human cells, the processing of the propeptide is an autocatalytic, multistep process proceeding from an inactive 41kDa pro-form, through a 30kDa intermediate form, to the 28kDa mature form. Tyr87P and Gly90P were identified as the two major endopeptidase cleavage sites, converting the 30kDa form into the mature 28kDa form. The level of processing differs significantly in different human cell lines. In monocyte-derived macrophages U937 and prostate cancer cells PC-3, the 28kDa form is predominant, whereas in osteoblasts HOS the processing from the 30kDa form to the 28kDa form is significantly lower. The aminopeptidase activity of the enzyme and its subcellular localization are independent of the product, however the 30kDa form was not secreted in HOS cells. The activity of the resulting cathepsin H in U937 cells was significantly lower than that in HOS cells, presumably due to the high levels of endogenous cysteine protease inhibitor cystatin F present specifically in this cell line. These results provide an insight into the dependence of human cathepsin H processing and regulation on cell type.

Discovery of highly potent, nonsteroidal 17ß-hydroxysteroid dehydrogenase type 1 inhibitors by virtual high-throughput screening.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyzes the formation of the potent proliferation-stimulating hormone estradiol, and it is thus involved in the development of hormone-dependent breast cancer. Due to its high substrate specificity and the known relationships between its overexpression and disease incidence, 17ß-HSD1 is considered an attractive target for drug development. Here, we have used structure-based virtual high-throughput screening to successfully identify potent nonsteroidal 17ß-HSD1 inhibitors. Computational screening of a drug-like database containing 13 million compounds identified hits with a 2-benzylidenebenzofuran-3(2H)-one scaffold that we show to be highly potent 17ß-HSD1 inhibitors. The most potent in the series, compound 1, showed an IC(50) of 45nM in our 17ß-HSD1 inhibition assay, and also showed good selectivity for 17ß-HSD1 over 17ß-HSD2.

Discovery and kinetic evaluation of 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as inhibitors of cathepsin B.

Cathepsin B is a lysosomal cysteine protease that has various physiological and pathophysiological functions. We present here the discovery of 6-substituted 4-benzylthio-1,3,5-triazin-2(1H)-ones as inhibitors of cathepsin B, starting from screening of a library of variously 2,4,6-trisubstituted 1,3,5-triazines and 1,3,5-triazin-2(1H)-ones on three different human cathepsins. The synthesis and enzymatic evaluation of a focused library of new 1,3,5-triazin-2(1H)-ones is also described. The detailed kinetics analyses have shown that these compounds can act as reversible, partial mixed-type inhibitors of cathepsin B, with K(i) and K(i)' values in the low micromolar range. The inhibitory activities of selected compounds were also assessed against two related cysteine proteases, cathepsin H and cathepsin L, to estimate their selectivity; these compounds have a selective profile for catB and catL over catH.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the ß(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated ß(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the ß(2) tail. This was shown by molecular modeling of the ß(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Small molecule inhibitors of peptidoglycan synthesis targeting the lipid II precursor.

Bacterial peptidoglycan glycosyltransferases (GTs) of family 51 catalyze the polymerization of the lipid II precursor into linear peptidoglycan strands. This activity is essential to bacteria and represents a validated target for the development of new antibacterials. Application of structure-based virtual screening to the National Cancer Institute library using eHits program and the structure of the glycosyltransferase domain of the Staphylococcus aureus penicillin-binding protein 2 resulted in the identification of two small molecules analogues 5, a 2-[1-[(2-chlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine and 5b, a 2-[1-[(3,4-dichlorophenyl)methyl]-2-methyl-5-methylsulfanylindol-3-yl]ethanamine that exhibit antibacterial activity against several Gram-positive bacteria but were less active on Gram-negative bacteria. The two compounds inhibit the activity of five GTs in the micromolar range. Investigation of the mechanism of action shows that the compounds specifically target peptidoglycan synthesis. Unexpectedly, despite the fact that the compounds were predicted to bind to the GT active site, compound 5b was found to interact with the lipid II substrate via the pyrophosphate motif. In addition, this compound showed a negatively charged phospholipid-dependent membrane depolarization and disruption activity. These small molecules are promising leads for the development of more active and specific compounds to target the essential GT step in cell wall synthesis.

Synthesis and biological evaluation of (6- and 7-phenyl) coumarin derivatives as selective nonsteroidal inhibitors of 17ß-hydroxysteroid dehydrogenase type 1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is an enzyme that catalyzes NADPH-dependent reduction of the weak estrogen, estrone, into the most potent estrogen, estradiol, which exerts proliferative effects via the estrogen receptors. Overexpression of 17ß-HSD1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis; thus, 17ß-HSD1 represents an attractive target for the development of new therapies. We have discovered that simple coumarines 1 and 2 significantly inhibit 17ß-HSD1 in a recombinant enzyme assay, with high selectivity against 17ß-HSD2. We postulated that the introduction of various p-substituted phenyl moieties to position 6 or 7 of the coumarin core using the Suzuki-Miyaura cross-coupling reaction would provide mimetics of steroidal structures with improved inhibition of 17ß-HSD1. The best inhibitor in the series proved to be 6a, with an IC(50) of 270 nM, and with exceptional selectivity for 17ß-HSD1 over 17ß-HSD2 and against the α and ß estrogen receptors.

Design and synthesis of substrate mimetics based on an indole scaffold: potential inhibitors of 17ß-HSD type 1.

BACKGROUND: Human 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) acts at a pre-receptor level. It catalyzes NADPH-dependent reduction of the weak estrogen estrone into the most potent estrogen estradiol, which exerts its proliferative effects via estrogen receptors. Overexpression of 17ß-HSD1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis. 17ß-HSD1 thus represents an attractive target for development of new drugs. METHODS: We designed and synthesized a series of 3-, 5- and 6-phenyl indole derivatives as mimetics of the steroid substrate estrone. All of these compounds were evaluated for inhibition of recombinant human 17ß-HSD1 from Escherichia coli, at concentrations of 0.6 µM and 6.0 µM. RESULTS: Among 14 indole derivatives, compound 9 was an initial hit inhibitor of 17ß-HSD1, with moderate inhibition (64% at 6 µM). Molecular docking into the crystal structure of 17ß-HSD1 (1A27) revealed that this 5-phenyl indole derivative binds to 17ß-HSD1 similarly to co-crystalized E2. Compound 9 forms two H-bonds with 17ß-HSD1: one between the indole nitrogen and His222, and the second between the phenolic OH group and catalytic Tyr155. CONCLUSIONS: The indole scaffold is one of the possible starting points for the design of substrate mimetics of the steroid substrate estrone. Our study shows that these 6- and, especially, 5-phenol indole derivatives can act as moderate inhibitors of 17ß-HSD1. Based on inhibition assays and docking simulations, we can infer further improvements of the 5-phenol indole derivatives that might result in better inhibition profiles.

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening.

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add L-Ala, D-Glu, meso-A(2)pm or L-Lys, and D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute 'Diversity Set' on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC(50)=10 microM) and one novel MurF inhibitor (IC(50)=63 microM).

Discovery of new inhibitors of aldo-keto reductase 1C1 by structure-based virtual screening.

Aldo-keto reductase 1C1 is a hydroxysteroid dehydrogenase that inactivates progesterone by converting it to 20alpha-hydroxyprogesterone. It also inactivates 3alpha,5alpha-tetrahydroprogesterone, an allosteric modulator of the gamma-aminobutyric acid receptor that has anaesthetic, analgesic, anxiolytic and anti-convulsant effects. Inhibitors of aldo-keto reductase 1C1 are thus very interesting as potential agents for the treatment of endometrial cancer, premenstrual syndrome, catamenial epilepsy, and depressive disorders, and for the maintenance of pregnancy. We have used the molecular docking program eHiTS for virtual screening of 1990 compounds from the National Cancer Institute "Diversity Set". Fifty compounds with the highest predicted binding energies were then evaluated in vitro. Three structurally diverse hits were obtained that inhibit aldo-keto reductase 1C1 in the low micromolar range of IC(50) values. These hits represent promising starting points for structural optimization in hit-to-lead development.