Evaluating the druggability of TrmD, a potential antibacterial target, through design and microbiological profiling of a series of potent TrmD inhibitors.

The post-transcriptional modifier tRNA-(N1G37) methyltransferase (TrmD) has been proposed to be essential for growth in many Gram-negative and Gram-positive pathogens, however previously reported inhibitors show only weak antibacterial activity. In this work, optimisation of fragment hits resulted in compounds with low nanomolar TrmD inhibition incorporating features designed to enhance bacterial permeability and covering a range of physicochemical space. The resulting lack of significant antibacterial activity suggests that whilst TrmD is highly ligandable, its essentiality and druggability are called into question.

Incorporating Target-Specific Pharmacophoric Information into Deep Generative Models for Fragment Elaboration.

Despite recent interest in deep generative models for scaffold elaboration, their applicability to fragment-to-lead campaigns has so far been limited. This is primarily due to their inability to account for local protein structure or a user's design hypothesis. We propose a novel method for fragment elaboration, STRIFE, that overcomes these issues. STRIFE takes as input fragment hotspot maps (FHMs) extracted from a protein target and processes them to provide meaningful and interpretable structural information to its generative model, which in turn is able to rapidly generate elaborations with complementary pharmacophores to the protein. In a large-scale evaluation, STRIFE outperforms existing, structure-unaware, fragment elaboration methods in proposing highly ligand-efficient elaborations. In addition to automatically extracting pharmacophoric information from a protein target's FHM, STRIFE optionally allows the user to specify their own design hypotheses.

Characterizing the binding and function of TARP γ8-selective AMPA receptor modulators.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)-type glutamate receptors (AMPARs) are the predominant excitatory neurotransmitter receptors in the brain, where they mediate synaptic transmission and plasticity. Excessive AMPAR activation leads to diseases such as epilepsy. AMPAR properties are modulated by auxiliary proteins and foremost by the transmembrane AMPAR regulatory proteins (TARPs). These distribute in unique expression patterns across the brain, rendering AMPAR/TARP complexes promising targets for region-specific therapeutic intervention. TARP γ8 is predominantly expressed in the forebrain and is enriched in the hippocampus, a region associated with temporal lobe epilepsy. Recent high-throughput medicinal chemistry screens have identified multiple promising compounds that selectively target AMPARs associated with γ8 and hold promise for epilepsy treatment. However, how these modulators target the receptor complex is currently unknown. Here, we use a combination of ligand docking, molecular dynamics simulations, and electrophysiology to address this question. We identify a conserved oxindole isostere, shared between three structurally diverse modulators (LY-3130481, JNJ-55511118, and JNJ-61432059) as the major module engaging γ8 by an H-bond to Asn-172 (γ8). The remaining variable region of each molecule likely targets the receptor complex in ligand-selective modes. Functional data reveal parallels in the underlying modulatory action of two prominent compounds. This work will aid development of refined AMPAR epilepsy therapeutics and facilitate to uncover the mechanisms by which TARPs modulate the receptor.

Discovery of potent and selective 5-azaindazole inhibitors of leucine-rich repeat kinase 2 (LRRK2) - Part 1.

Parkinson's disease is a relatively common neurological disorder with incidence increasing with age. Present treatments merely alleviate the symptoms and do not alter the course of the disease, thus identification of disease modifying therapies represents a significant unmet medical need. Mutations in the LRRK2 gene are risk-factors for developing PD and it has been hypothesized that the increased kinase activity of certain LRRK2 mutants are responsible for the damage of the dopaminergic neurons, thus LRRK2 inhibitors offer the potential to target an underlying cause of the disease. In this communication, we describe hit-to-lead medicinal chemistry program on a novel series of 5-azaindazoles. Compound 1, obtained from high-throughput screening was optimized to a highly potent, selective series of molecules with promising DMPK properties. Introduction of heterocycles at the 3-position were found to significantly increase the potency and kinase selectivity, whilst changes to the 4-chlorobenzyl group improved the physicochemical properties. Our series was licensed to a major pharmaceutical company for further development.

Potent inhibitors of malarial P. Falciparum protein kinase G: Improving the cell activity of a series of imidazopyridines.

Development of a class of bicyclic inhibitors of the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG), starting from known compounds with activity against a related parasite PKG orthologue, is reported. Examination of key sub-structural elements led to new compounds with good levels of inhibitory activity against the recombinant kinase and in vitro activity against the parasite. Key examples were shown to possess encouraging in vitro ADME properties, and computational analysis provided valuable insight into the origins of the observed activity profiles.

Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency, selectivity and structural novelty.

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.

Trisubstituted thiazoles as potent and selective inhibitors of Plasmodium falciparum protein kinase G (PfPKG).

A series of trisubstituted thiazoles have been identified as potent inhibitors of Plasmodium falciparum (Pf) cGMP-dependent protein kinase (PfPKG) through template hopping from known Eimeria PKG (EtPKG) inhibitors. The thiazole series has yielded compounds with improved potency, kinase selectivity and good in vitro ADME properties. These compounds could be useful tools in the development of new anti-malarial drugs in the fight against drug resistant malaria.

Biochemical and antiparasitic properties of inhibitors of the Plasmodium falciparum calcium-dependent protein kinase PfCDPK1.

PfCDPK1 is a Plasmodium falciparum calcium-dependent protein kinase, which has been identified as a potential target for novel antimalarial chemotherapeutics. In order to further investigate the role of PfCDPK1, we established a high-throughput in vitro biochemical assay and used it to screen a library of over 35,000 small molecules. Five chemical series of inhibitors were initially identified from the screen, from which series 1 and 2 were selected for chemical optimization. Indicative of their mechanism of action, enzyme inhibition by these compounds was found to be sensitive to both the ATP concentration and substitution of the amino acid residue present at the "gatekeeper" position at the ATP-binding site of the enzyme. Medicinal chemistry efforts led to a series of PfCDPK1 inhibitors with 50% inhibitory concentrations (IC50s) below 10 nM against PfCDPK1 in a biochemical assay and 50% effective concentrations (EC50s) less than 100 nM for inhibition of parasite growth in vitro. Potent inhibition was combined with acceptable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and equipotent inhibition of Plasmodium vivax CDPK1. However, we were unable to correlate biochemical inhibition with parasite growth inhibition for this series overall. Inhibition of Plasmodium berghei CDPK1 correlated well with PfCDPK1 inhibition, enabling progression of a set of compounds to in vivo evaluation in the P. berghei rodent model for malaria. These chemical series have potential for further development as inhibitors of CDPK1.

The future of machine learning for small-molecule drug discovery will be driven by data.

Many studies have prophesied that the integration of machine learning techniques into small-molecule therapeutics development will help to deliver a true leap forward in drug discovery. However, increasingly advanced algorithms and novel architectures have not always yielded substantial improvements in results. In this Perspective, we propose that a greater focus on the data for training and benchmarking these models is more likely to drive future improvement, and explore avenues for future research and strategies to address these data challenges.

Targeted protein degradation in CNS disorders: a promising route to novel therapeutics?

Targeted protein degradation (TPD) is a rapidly expanding field, with various PROTACs (proteolysis-targeting chimeras) in clinical trials and molecular glues such as immunomodulatory imide drugs (IMiDs) already well established in the treatment of certain blood cancers. Many current approaches are focused on oncology targets, leaving numerous potential applications underexplored. Targeting proteins for degradation offers a novel therapeutic route for targets whose inhibition remains challenging, such as protein aggregates in neurodegenerative diseases. This mini review focuses on the prospect of utilizing TPD for neurodegenerative disease targets, particularly PROTAC and molecular glue formats and opportunities for novel CNS E3 ligases. Some key challenges of utilizing such modalities including molecular design of degrader molecules, drug delivery and blood brain barrier penetrance will be discussed.

Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1).

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.

Identification of novel p-cresol inhibitors that reduce Clostridioides difficile's ability to compete with species of the gut microbiome.

Treatment of Clostridioides difficile infection (CDI) is expensive and complex, with a high proportion of patients suffering infection relapse (20-35%), and some having multiple relapses. A healthy, unperturbed gut microbiome provides colonisation resistance against CDI through competition for nutrients and space. However, antibiotic consumption can disturb the gut microbiota (dysbiosis) resulting in the loss of colonisation resistance allowing C. difficile to colonise and establish infection. A unique feature of C. difficile is the production of high concentrations of the antimicrobial compound para-cresol, which provides the bacterium with a competitive advantage over other bacteria found in the gut. p-cresol is produced by the conversion of para-Hydroxyphenylacetic acid (p-HPA) by the HpdBCA enzyme complex. In this study, we have identified several promising inhibitors of HpdBCA decarboxylase, which reduce p-cresol production and render C. difficile less able to compete with a gut dwelling Escherichia coli strain. We demonstrate that the lead compound, 4-Hydroxyphenylacetonitrile, reduced p-cresol production by 99.0 ± 0.4%, whereas 4-Hydroxyphenylacetamide, a previously identified inhibitor of HpdBCA decarboxylase, only reduced p-cresol production by 54.9 ± 13.5%. To interpret efficacy of these first-generation inhibitors, we undertook molecular docking studies that predict the binding mode for these compounds. Notably, the predicted binding energy correlated well with the experimentally determined level of inhibition, providing a molecular basis for the differences in efficacy between the compounds. This study has identified promising p-cresol production inhibitors whose development could lead to beneficial therapeutics that help to restore colonisation resistance and therefore reduce the likelihood of CDI relapse.

Synthesis and structure-activity relationships of a novel series of pyrimidines as potent inhibitors of TBK1/IKKε kinases.

The design, synthesis and structure-activity relationships of a novel series of 2,4-diamino-5-cyclopropyl pyrimidines is described. Starting from BX795, originally reported to be a potent inhibitor of PDK1, we have developed compounds with improved selectivity and drug-like properties. These compounds have been evaluated in a range of cellular and in vivo assays, enabling us to probe the putative role of the TBK1/IKKε pathway in inflammatory diseases.

Fragment-based target screening as an empirical approach to prioritising targets: a case study on antibacterials.

Here, we describe a novel workflow combining informatic and experimental approaches to enable evidence-based prioritising of targets from large sets in parallel. High-throughput protein production and biophysical fragment screening is used to identify those targets that are tractable and ligandable. As proof of concept we have applied this to a set of antibacterial targets comprising 146 essential genes. Of these targets, 51 were selected and 38 delivered results that allowed us to rank them by ligandability. The data obtained against these derisked targets have enabled rapid progression into structurally enabled drug discovery projects, demonstrating the practical value of the fragment-based target screening workflow.

Design of the LifeArc Index Set and Retrospective Review of Its Performance: A Collection for Sharing.

Building, curating, and maintaining a compound collection is an expensive operation, beyond the scope of most academic organizations. Here we describe the selection criteria used to compile the LifeArc diversity set from commercial suppliers and the process we undertook to generate our representative LifeArc index set. The aim was to avoid a "junk in, junk out" screen collection to increase chemical tractability going forward, while maximizing diversity. Using historical LifeArc screening data, we demonstrate that the index set was predictive of ligandability and that progressable hits could be identified by mining associated clusters within our larger diversity set. Indeed, a higher percentage of index-derived hit clusters were found to have been progressed into hit-to-lead programs, reflecting better drug-likeness. In practice, the library has been shared widely with academic groups and used routinely within LifeArc to assess the ligandability of novel targets. Its small size is well suited to meet the needs of medium-throughput screening in labs with either limited automation, limited precious or expensive reagents, or complex cellular assays. The strategy of screening a small set in combination with rapid hit analog follow-up has demonstrated the utility of finding active clusters for potential development against challenging targets.

Design, Synthesis, and Testing of Potent, Selective Hepsin Inhibitors via Application of an Automated Closed-Loop Optimization Platform.

Hepsin is a membrane-anchored serine protease whose role in hepatocyte growth factor (HGF) signaling and epithelial integrity makes it a target of therapeutic interest in carcinogenesis and metastasis. Using an integrated design, synthesis, and screening platform, we were able to rapidly develop potent and selective inhibitors of hepsin. In progressing from the initial hit 7 to compound 53, the IC50 value against hepsin was improved from ∼1 µM to 22 nM, and the selectivity over urokinase-type plasminogen activator (uPA) was increased from 30-fold to >6000-fold. Subsequent in vitro ADMET profiling and cellular studies confirmed that the leading compounds are useful tools for interrogating the role of hepsin in breast tumorigenesis.

Characterization of Three Druggable Hot-Spots in the Aurora-A/TPX2 Interaction Using Biochemical, Biophysical, and Fragment-Based Approaches.

The mitotic kinase Aurora-A and its partner protein TPX2 (Targeting Protein for Xenopus kinesin-like protein 2) are overexpressed in cancers, and it has been proposed that they work together as an oncogenic holoenzyme. TPX2 is responsible for activating Aurora-A during mitosis, ensuring proper cell division. Disruption of the interface with TPX2 is therefore a potential target for novel anticancer drugs that exploit the increased sensitivity of cancer cells to mitotic stress. Here, we investigate the interface using coprecipitation assays and isothermal titration calorimetry to quantify the energetic contribution of individual residues of TPX2. Residues Tyr8, Tyr10, Phe16, and Trp34 of TPX2 are shown to be crucial for robust complex formation, suggesting that the interaction could be abrogated through blocking any of the three pockets on Aurora-A that complement these residues. Phosphorylation of Aurora-A on Thr288 is also necessary for high-affinity binding, and here we identify arginine residues that communicate the phosphorylation of Thr288 to the TPX2 binding site. With these findings in mind, we conducted a high-throughput X-ray crystallography-based screen of 1255 fragments against Aurora-A and identified 59 hits. Over three-quarters of these hits bound to the pockets described above, both validating our identification of hotspots and demonstrating the druggability of this protein-protein interaction. Our study exemplifies the potential of high-throughput crystallography facilities such as XChem to aid drug discovery. These results will accelerate the development of chemical inhibitors of the Aurora-A/TPX2 interaction.

The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover.

Increasing evidence implicates serine proteinases in the proteolytic cascades leading to the pathological destruction of extracellular matrices such as cartilage in osteoarthritis (OA). We have previously demonstrated that the type II transmembrane serine proteinase (TTSP) matriptase acts as a novel initiator of cartilage destruction via the induction and activation of matrix metalloproteinases (MMPs). Hepsin is another TTSP expressed in OA cartilage such that we hypothesized this proteinase may also contribute to matrix turnover. Herein, we demonstrate that addition of hepsin to OA cartilage in explant culture induced significant collagen and aggrecan release and activated proMMP-1 and proMMP-3. Furthermore, hepsin directly cleaved the aggrecan core protein at a novel cleavage site within the interglobular domain. Hepsin expression correlated with synovitis as well as tumour necrosis factor α expression, and was induced in cartilage by a pro-inflammatory stimulus. However, a major difference compared to matriptase was that hepsin demonstrated markedly reduced capacity to activate proteinase-activated receptor-2. Overall, our data suggest that hepsin, like matriptase, induces potent destruction of the extracellular matrix whilst displaying distinct efficiencies for the cleavage of specific substrates.

A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission.

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.Protein kinases are promising drug targets for treatment of malaria. Here, starting with a medicinal chemistry approach, Baker et al. generate an imidazopyridine that selectively targets Plasmodium falciparum PKG, inhibits blood stage parasite growth in vitro and in mice and blocks transmission to mosquitoes.

Competitive binding of a benzimidazole to the histone-binding pocket of the Pygo PHD finger.

The Pygo-BCL9 complex is a chromatin reader, facilitating ß-catenin-mediated oncogenesis, and is thus emerging as a potential therapeutic target for cancer. Its function relies on two ligand-binding surfaces of Pygo's PHD finger that anchor the histone H3 tail methylated at lysine 4 (H3K4me) with assistance from the BCL9 HD1 domain. Here, we report the first use of fragment-based screening by NMR to identify small molecules that block protein-protein interactions by a PHD finger. This led to the discovery of a set of benzothiazoles that bind to a cleft emanating from the PHD-HD1 interface, as defined by X-ray crystallography. Furthermore, we discovered a benzimidazole that docks into the H3K4me specificity pocket and displaces the native H3K4me peptide from the PHD finger. Our study demonstrates the ligandability of the Pygo-BCL9 complex and uncovers a privileged scaffold as a template for future development of lead inhibitors of oncogenesis.