Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1

Genetic studies indicate that the enzyme pteridine reductase?1 (PTR1) is essential for the survival of the protozoan parasite Trypanosoma brucei. Herein, we describe the development and optimisation of a novel series of PTR1 inhibitors, based on benzo[d]imidazol-2-amine derivatives. Data are reported on 33 compounds. This series was initially discovered by a virtual screening campaign (J. Med. Chem., 2009, 52, 4454). The inhibitors adopted an alternative binding mode to those of the natural ligands, biopterin and dihydrobiopterin, and classical inhibitors, such as methotrexate. Using both rational medicinal chemistry and structure-based approaches, we were able to derive compounds with potent activity against T.?brucei PTR1 (K(i)(app)=7?nM), which had high selectivity over both human and T.?brucei dihydrofolate reductase. Unfortunately, these compounds displayed weak activity against the parasites. Kinetic studies and analysis indicate that the main reason for the lack of cell potency is due to the compounds having insufficient potency against the enzyme, which can be seen from the low K(m) to K(i) ratio (K(m)=25?nM and K(i)=2.3?nM, respectively).     

Identification and Structure–Activity Relationship Studies of Small‐Molecule Inhibitors of the Methyllysine Reader Protein Spindlin1

The methyllysine reader protein Spindlin1 has been implicated in the tumorigenesis of several types of cancer and may be an attractive novel therapeutic target. Small‐molecule inhibitors of Spindlin1 should be valuable as chemical probes as well as potential new therapeutics. We applied an iterative virtual screening campaign, encompassing structure‐ and ligand‐based approaches, to identify potential Spindlin1 inhibitors from databases of commercially available compounds. Our in silico studies coupled with in vitro testing were successful in identifying novel Spindlin1 inhibitors. Several 4‐aminoquinazoline and quinazolinethione derivatives were among the active hit compounds, which indicated that these scaffolds represent promising lead structures for the development of Spindlin1 inhibitors. Subsequent lead optimization studies were hence carried out, and numerous derivatives of both lead scaffolds were synthesized. This resulted in the discovery of novel inhibitors of Spindlin1 and helped explore the structure–activity relationships of these inhibitor series.     

Discovery and Characterization of Multiple Classes of Human CatSper Blockers

The cation channel of sperm (CatSper) is a validated target for nonhormonal male contraception, but it lacks selective blockers, hindering studies to establish its role in both motility and capacitation. Via an innovative calcium uptake assay utilizing human sperm we discovered novel inhibitors of CatSper function from a high-throughput screening campaign of 72,000 compounds. Preliminary SAR was established for seven hit series. HTS hits or their more potent analogs blocked potassium-induced depolarization and noncompetitively inhibited progesterone-induced CatSper activation. CatSper channel blockade was confirmed by patch clamp electrophysiology and these compounds inhibited progesterone- and prostaglandin E1-induced hyperactivated sperm motility. One of the hit compounds is a potent CatSper inhibitor with high selectivity for CatSper over hCav1.2, hNav1.5, moderate selectivity over hSlo3 and hERG, and low cytotoxicity and is therefore the most promising inhibitor identified in this study. These new CatSper blockers serve as useful starting points for chemical probe development and drug discovery efforts.     

Structure-Activity Relationship and Mode-Of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors

CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with antimycobacterial activity. Herein, we report the screening of a similarity-oriented library based on the former hit compound, the evaluation of affinity toward CYP121, and activity against M. bovis BCG. The results enabled a comprehensive SAR study, which was extended through the synthesis of promising compounds and led to the identification of favorable features for affinity and/or activity and hit compounds with 2.7-fold improved potency. Mode of action studies show that the hit compounds inhibit substrate conversion and highlighted CYP121 as the main antimycobacterial target of our compounds. Exemplified complex crystal structures of CYP121 with three inhibitors reveal a common binding site. Engaging in both hydrophobic interactions as well as hydrogen bonding to the sixth iron ligand, our compounds block a solvent channel leading to the active site heme. Additionally, we report the first CYP inhibitors that are able to reduce the intracellular replication of M. bovis BCG in macrophages, emphasizing their potential as future drug candidates against TB.     

Investigation of Trypanothione Reductase as a Drug Target in Trypanosoma brucei

There is an urgent need for new drugs for the treatment of tropical parasitic diseases such as human African trypanosomiasis, which is caused by Trypanosoma brucei. The enzyme trypanothione reductase (TryR) is a potential drug target within these organisms. Herein we report the screening of a 62 000 compound library against T. brucei TryR. Further work was undertaken to optimise potency and selectivity of two novel‐compound series arising from the enzymatic and whole parasite screens and mammalian cell counterscreens. Both of these series, containing either a quinoline or pyrimidinopyrazine scaffold, yielded low micromolar inhibitors of the enzyme and growth of the parasite. The challenges of inhibiting TryR with druglike molecules is discussed.     

Delineation of the molecular mechanisms of nucleoside recognition by cytidine deaminase through virtual screening

Cytidine deaminase (EC 3.5.4.5, CDA), an enzyme of the pyrimidine salvage pathways, is responsible for the degradation and inactivation of several cytidine‐based antitumor drugs such as cytarabine, gemcitabine, decitabine, and azacytidine. Thus, CDA inhibitors are highly sought after as compounds to be co‐administered with said drugs to improve their effectiveness. Alternatively, the design of antitumor drugs not susceptible to the action of CDA is also regarded as an attractive solution. Herein we describe a virtual screen for CDA ligands based on chemical similarity and molecular docking. The campaign led to the identification of three novel inhibitors and one novel substrate, with a 19 % hit rate, and allowed a significant extension of the structure–activity relationships, also in light of the compounds that resulted inactive. The most active compound identified through the screen is the inhibitor pseudoisocytidine, which has the potential to serve as a lead for highly stable compounds. The study also delineated the detrimental effect of 5‐aza and 6‐aza substitutions, the incompatibility of the presence of an amino group at the 3′‐position, as well as the presence of very strict steric requirements around the 2′‐arabino position and, even more, the N4‐position. Importantly, these features can be exploited for the design of novel anti‐neoplastic agents resistant to the action of CDA.     

Identification of inhibitors of the Leishmania cdc2-related protein kinase CRK3

New drugs are urgently needed for the treatment of tropical parasitic diseases such as leishmaniasis and human African trypanosomiasis (HAT). This work involved a high-throughput screen of a focussed kinase set of ~3400 compounds to identify potent and parasite-selective inhibitors of an enzymatic Leishmania CRK3-cyclin 6 complex. The aim of this study is to provide chemical validation that Leishmania CRK3-CYC6 is a drug target. Eight hit series were identified, of which four were followed up. The optimisation of these series using classical SAR studies afforded low-nanomolar CRK3 inhibitors with significant selectivity over the closely related human cyclin dependent kinase CDK2.     

3‐Aminoazetidin‐2‐one Derivatives as N‐Acylethanolamine Acid Amidase (NAAA) Inhibitors Suitable for Systemic Administration

N‐Acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide (PEA). PEA has been shown to exert anti‐inflammatory and antinociceptive effects in animals by engaging peroxisome proliferator‐activated receptor α (PPAR‐α). Thus, preventing PEA degradation by inhibiting NAAA may provide a novel approach for the treatment of pain and inflammatory states. Recently, 3‐aminooxetan‐2‐one compounds were identified as a class of highly potent NAAA inhibitors. The utility of these compounds is limited, however, by their low chemical and plasma stabilities. In the present study, we synthesized and tested a series of N‐(2‐oxoazetidin‐3‐yl)amides as a novel class of NAAA inhibitors with good potency and improved physicochemical properties, suitable for systemic administration. Moreover, we elucidated the main structural features of 3‐aminoazetidin‐2‐one derivatives that are critical for NAAA inhibition.     

Exploring acyclic nucleoside analogues as inhibitors of Mycobacterium tuberculosis thymidylate kinase

In the search for novel inhibitors of the enzyme thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt), an attractive target for novel antituberculosis agents, we report herein the discovery of the first acyclic nucleoside analogues that potently and selectively inhibit TMPKmt. The most potent compounds in this series are (Z)-butenylthymines carrying a naphtholactam or naphthosultam moiety at position 4, which display K(i) values of 0.42 and 0.27 microM, respectively. Docking studies followed by molecular dynamics simulations performed to rationalize the interaction of this new family of inhibitors with the target enzyme revealed a key interaction between the distal substituent and Arg 95 in the target enzyme. The fact that these inhibitors are more easily synthesizable than previously identified TMPKmt inhibitors, together with their potency against the target enzyme, makes them attractive lead compounds for further optimization.     

Synthesis and evaluation of indatraline-based inhibitors for trypanothione reductase

The search for novel compounds of relevance to the treatment of diseases caused by trypanosomatid protozoan parasites continues. Screening of a large library of known bioactive compounds has led to several drug-like starting points for further optimisation. In this study, novel analogues of the monoamine uptake inhibitor indatraline were prepared and assessed both as inhibitors of trypanothione reductase (TryR) and against the parasite Trypanosoma brucei. Although it proved difficult to significantly increase the potency of the original compound as an inhibitor of TryR, some insight into the preferred substituent on the amine group and in the two aromatic rings of the parent indatraline was deduced. In addition, detailed mode of action studies indicated that two of the inhibitors exhibit a mixed mode of inhibition.     

Design,Synthesis and Evaluation of Triazole‐Pyrimidine Analogues as SecA Inhibitors

SecA, a key component of the bacterial Sec‐dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. Through a combination of virtual screening and experimental exploration of the surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA‐21, and studied a series of analogues by systematic dissections of the core scaffold. Evaluation of these analogues allowed us to establish an initial structure–activity relationship in SecA inhibition. The best compounds in this group are potent inhibitors of SecA‐dependent protein‐conducting channel activity and protein translocation activity at low‐ to sub‐micromolar concentrations. They also have minimal inhibitory concentration (MIC) values against various strains of bacteria that correlate well with the SecA and protein translocation inhibition data. These compounds are effective against methicillin‐resistant Staphylococcus aureus strains with various levels of efflux pump activity, indicating the capacity of SecA inhibitors to null the effect of multidrug resistance. Results from studies of drug‐affinity‐responsive target stability and protein pull‐down assays are consistent with SecA as a target for these compounds.     

Three-dimensional pharmacophore design and biochemical screening identifies substituted 1,2,4-triazoles as inhibitors of the annexin A2-S100A10 protein interaction

Protein interactions are increasingly appreciated as targets in small‐molecule drug discovery. The interaction between the adapter protein S100A10 and its binding partner annexin A2 is a potentially important drug target. To obtain small‐molecule starting points for inhibitors of this interaction, a three‐dimensional pharmacophore model was constructed from the X‐ray crystal structure of the complex between S100A10 and annexin A2. The pharmacophore model represents the favourable hydrophobic and hydrogen bond interactions between the two partners, as well as spatial and receptor site constraints (excluded volume spheres). Using this pharmacophore model, UNITY flex searches were carried out on a 3D library of 0.7 million commercially available compounds. This resulted in 568 hit compounds. Subsequently, GOLD docking studies were performed on these hits, and a set of 190 compounds were purchased and tested biochemically for inhibition of the protein interaction. Three compounds of similar chemical structure were identified as genuine inhibitors of the binding of annexin A2 to S100A10. The binding modes predicted by GOLD were in good agreement with their UNITY‐generated conformations. We synthesised a series of analogues revealing areas critical for binding. Thus computational predictions and biochemical screening can be used successfully to derive novel chemical classes of protein–protein interaction blockers.     

Identification of New KRAS G12D Inhibitors through Computer-Aided Drug Discovery Methods

Owing to several mutations, the oncogene Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) is activated in the majority of cancers, and targeting it has been pharmacologically challenging. In this study, using an in silico approach comprised of pharmacophore modeling, molecular docking, and molecular dynamics simulations, potential KRAS G12D inhibitors were investigated. A ligand-based common feature pharmacophore model was generated to identify the framework necessary for effective KRAS inhibition. The chemical features in the selected pharmacophore model comprised two hydrogen bond donors, one hydrogen bond acceptor, two aromatic rings and one hydrophobic feature. This model was used for screening in excess of 214,000 compounds from InterBioScreen (IBS) and ZINC databases. Eighteen compounds from the IBS and ten from the ZINC database mapped onto the pharmacophore model and were subjected to molecular docking. Molecular docking results highlighted a higher affinity of four hit compounds towards KRAS G12D in comparison to the reference inhibitor, BI-2852. Sequential molecular dynamics (MD) simulation studies revealed all four hit compounds them possess higher KRAS G12D binding free energy and demonstrate stable polar interaction with key residues. Further, Principal Component Analysis (PCA) analysis of the hit compounds in complex with KRAS G12D also indicated stability. Overall, the research undertaken provides strong support for further in vitro testing of these newly identified KRAS G12D inhibitors, particularly Hit1 and Hit2.     

Designed amyloid beta peptide fibril - a tool for high-throughput screening of fibril inhibitors

Amyloid beta peptide (Abeta) fibril formation is widely believed to be the causative event of Alzheimer's disease pathogenesis. Therapeutic approaches are therefore in development that target various sites in the production and aggregation of Abeta. Herein we present a high-throughput screening tool to generate novel hit compounds that block Abeta fibril formation. This tool is an application for our fibril model (Abeta(16-37)Y(20)K(22)K(24))(4), which is a covalent assembly of four Abeta fragments. With this tool, screening studies are complete within one hour, as opposed to days with native Abeta(1-40). A Z' factor of 0.84+/-0.03 was determined for fibril formation and inhibition, followed by the reporter molecule thioflavin T. Herein we also describe the analysis of a broad range of reported inhibitors and non-inhibitors of Abeta fibril formation to test the validity of the system.     

A Series of 2-((1-Phenyl-1H-imidazol-5-yl)methyl)-1H-indoles as Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors

Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising therapeutic target in cancer immunotherapy and neurological disease. Thus, searching for highly active inhibitors for use in human cancers is now a focus of widespread research and development efforts. In this study, we report the structure-based design of 2-(5-imidazolyl)indole derivatives, a series of novel IDO1 inhibitors which have been designed and synthesized based on our previous study using N1-substituted 5-indoleimidazoles. Among these, we have identified one with a strong IDO1 inhibitory activity (IC₅₀=0.16 μM, EC₅₀=0.3 μM). Structural-activity relationship (SAR) and computational docking simulations suggest that a hydroxyl group favorably interacts with a proximal Ser167 residue in Pocket A, improving IDO1 inhibitory potency. The brain penetrance of potent compounds was estimated by calculation of the Blood Brain Barrier (BBB) Score and Brain Exposure Efficiency (BEE) Score. Many compounds had favorable scores and the two most promising compounds were advanced to a pharmacokinetic study which demonstrated that both compounds were brain penetrant. We have thus discovered a flexible scaffold for brain penetrant IDO1 inhibitors, exemplified by several potent, brain penetrant, agents. With this promising scaffold, we provide herein a basis for further development of brain penetrant IDO1 inhibitors.     

Design, synthesis, and biological activity of urea derivatives as anaplastic lymphoma kinase inhibitors

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells.     

Design,Synthesis and Evaluation of a Series of 1,5-Diaryl-1,2,3-triazole-4-carbohydrazones as Inhibitors of the YAP-TAZ/TEAD Complex

Starting from our previously reported hit, a series of 1,5-diaryl-1,2,3-triazole-4-carbohydrazones were synthesized and evaluated as inhibitors of the YAP/TAZ-TEAD complex. Their binding to hTEAD2 was confirmed by nanodifferential scanning fluorimetry, and some of the compounds were also found to moderately disrupt the YAP-TEAD interaction, as assessed by a fluorescence polarization assay. A TEAD luciferase gene reporter assay performed in HEK293T cells and RTqPCR measurements in MDA-MB231 cells showed that these compounds inhibit YAP/TAZ-TEAD activity to cells in the micromolar range. In spite of the cytotoxic effects displayed by some of the compounds of this series, they are still good starting points and can be suitably modified into an effective and viable YAP-TEAD disruptor in the future.     

Tertiary‐Amine‐Based Inhibitors of the Astacin Protease Meprin α

Metalloproteinases of the astacin family are drawing ever increasing attention as potential drug targets. However, knowledge regarding inhibitors thereof is limited in most cases. Crucial for the development of metalloprotease inhibitors is high selectivity, to avoid side effects brought about by inhibition of off‐target proteases and interference with physiological pathways. In this study we aimed at the design of novel selective inhibitors for the astacin proteinase meprin α. Based on a recently identified tertiary amine scaffold, a series of compounds was synthesized and evaluated. The compounds exhibit reasonable inhibitory activity with high selectivity over other metalloproteases. The isoenzyme meprin β is only slightly inhibited. Hence, the present study revealed a novel class of selective meprin α inhibitors with improved selectivity over known compounds.     

Discovery of Mono‐ and Disubstituted 1H‐Pyrazolo[3,4]pyrimidines and 9H‐Purines as Catalytic Inhibitors of Human DNA Topoisomerase IIα

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Based on structural data regarding the binding mode of AMP‐PNP (5′‐adenylyl‐β,γ‐imidodiphosphate) to htIIα, we designed a two‐stage virtual screening campaign that combines structure‐based pharmacophores and molecular docking. In the first stage, we identified several monosubstituted 9H‐purine compounds and a novel class of 1H‐pyrazolo[3,4]pyrimidines as inhibitors of htIIα. In the second stage, disubstituted analogues with improved cellular activities were discovered. Compounds from both classes were shown to inhibit htIIα‐mediated DNA decatenation, and surface plasmon resonance (SPR) experiments confirmed binding of these two compounds on the htIIα ATPase domain. Proposed complexes and interaction patterns between both compounds and htIIα were further analyzed in molecular dynamics simulations. Two compounds identified in the second stage showed promising anticancer activities in hepatocellular carcinoma (HepG2) and breast cancer (MCF‐7) cell lines. The discovered compounds are suitable starting points for further hit‐to‐lead development in anticancer drug discovery.     

Monitoring Binding of HIV‐1 Capsid Assembly Inhibitors Using 19F Ligand‐and 15N Protein‐Based NMR and X‐ray Crystallography: Early Hit Validation of a Benzodiazepine Series

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)‐1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high‐throughput screening using an in vitro capsid assembly assay (CAA). Here, we demonstrate how a combination of NMR and X‐ray co‐crystallography allowed for the rapid characterization of the early hits from this inhibitor series. Ligand‐based ¹⁹F NMR was used to confirm inhibitor binding specificity and reversibility as well as to identify the N‐terminal domain of the capsid (CA_NTD) as its molecular target. Protein‐based NMR (¹H and ¹⁵N chemical shift perturbation analysis) identified key residues within the CA_NTD involved in inhibitor binding, while X‐ray co‐crystallography confirmed the inhibitor binding site and its binding mode. Based on these results, two conformationally restricted cyclic inhibitors were designed to further validate the possible binding modes. These studies were crucial to early hit confirmation and subsequent lead optimization.