GPR84 is a proinflammatory G-protein-coupled receptor implicated in autoimmune and fibrotic disorders. Although orthosteric antagonists have been reported, their physicochemical limitations have hindered development. Here, we describe the discovery and optimization of a chromenopyrrole scaffold as a new class of orthosteric GPR84 antagonists. Guided by molecular modeling and iterative SAR, we identified ligands that competitively inhibit agonist binding, confirmed by Schild analysis and radioligand displacement. Structural refinement defined key steric and hydrophobic features required for high-affinity binding, culminating in the isolation of a single active enantiomer, 42E2 (pA 2 = 8.41, pK i = 8.16). This chemotype displays improved drug-like properties relative to earlier series and strong selectivity over related free fatty acid receptors. Preliminary pharmacokinetic studies indicate favorable solubility and plasma protein binding, although metabolic stability remains to be optimized. These results expand the chemical space for GPR84 modulation and provide a foundation for therapeutic development and mechanistic investigation.

Cyclophilins have been implicated in the pathophysiology of metabolic dysfunction-associated steatohepatitis (MASH). Pharmacological inhibition of the cyclophilin B isoform has the potential to attenuate liver fibrosis in MASH, but current cyclophilin inhibitors in clinical trials lack isoform selectivity. We previously reported the novel tri-vector small-molecule inhibitor 1 that exhibited improved subtype selectivity by simultaneously engaging three pockets on the surface of cyclophilins. Here, we present structure-activity relationships that address genotoxicity concerns, enhance subtype selectivity, improve pharmaceutical properties, and demonstrate strong efficacy in a MASH cellular model. Lead compound 11 is a potent cyclophilin B inhibitor with an encouraging pharmacokinetic profile suitable for further development.

In this study, the first industrial-scale high-throughput screening of nearly 350,000 drug-like molecules targeting the enzyme 17β-HSD10, a promising therapeutic target for Alzheimer's disease and cancers, is presented. Two novel series of potent 17β-HSD10 inhibitors that demonstrate low nanomolar potency against both the enzyme and in vivo cellular assays with minimal cytotoxicity were identified. These inhibitors were characterized further through a series of assays demonstrating ligand-protein interactions and co-crystallography, revealing un-/non-competitive inhibition with respect to the cofactor NADH, unlike previously published inhibitors. This work significantly advances the development of 17β-HSD10-targeting therapeutics, offering new potential leads for treating Alzheimer's disease and cancers.

The three human SNM1 metallo-β-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

G-protein-coupled receptor 84 (GPR84) is a proinflammatory orphan G-protein-coupled receptor implicated in several inflammatory and fibrotic diseases. Several agonist and antagonist ligands have been developed that target GPR84; however, a noncompetitive receptor blocker that was progressed to phase II clinical trials failed to demonstrate efficacy. New high-quality antagonists are required to investigate the pathophysiological role of GPR84 and to validate GPR84 as a therapeutic target. We previously reported the discovery of a novel triazine GPR84 competitive antagonist 1. Here, we describe an extensive structure-activity relationship (SAR) of antagonist 1 and also present in silico docking with supporting mutagenesis studies that reveals a potential binding pose for this type of orthosteric antagonist. Lead compound 42 is a potent GPR84 antagonist with a favorable pharmacokinetic (PK) profile suitable for further drug development.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

The role of the androgen receptor (AR) in the progression of prostate cancer (PCa) is well established and competitive inhibition of AR ligand binding domain (LBD) has been the mainstay of antiandrogen therapies for advanced and metastatic disease. However, the efficacy of such drugs is often limited by the emergence of resistance, mediated through point mutations and receptor splice variants lacking the AR-LBD. As a result, the prognosis for patients with malignant, castrate-resistant disease remains poor. The amino terminal domain (NTD) of the AR has been shown to be critical for AR function. Its modular activation function (AF-1) is important for both gene regulation and participation in protein-protein interactions. However, due to the intrinsically disordered structure of the domain, its potential as a candidate for therapeutic intervention has been generally overlooked. In this article, we describe the design and development of a functional cell-based assay aimed at identifying small-molecule inhibitors of the AR-NTD. We demonstrate the suitability of the assay for high-throughput screening platforms and validate two initial hits emerging from a small, targeted, library screen in PCa cells.

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-β-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential β-lactamase stable β-lactam mimics. Subsequent structure–activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL–carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models. The efficacy of carbapenem antibiotics can be compromised by metallo-β-lactamases, but a high-throughput screen followed by optimization has now enabled the discovery of indole-2-carboxylates (InCs) as potent broad-spectrum metallo-β-lactamase inhibitors. The results highlight the potential of InC–carbapenem combinations for clinical use as well as mechanism-guided approaches to combatting globally disseminated antibiotic resistant mechanisms.

The European Lead Factory combines assets and experience from major pharma with innovation and agility of academia and SMEs in a collaborative platform to expand access to high-throughput screening. With many successes heading towards the clinic, the organization is broadening its approach to screening and partnering. The European Lead Factory combines assets and experience from major pharma with innovation and agility of academia and SMEs in a collaborative platform to expand access to high-throughput screening. With many successes heading towards the clinic, the organization is broadening its approach to screening and partnering.

GPR84 is a poorly characterized, nominally orphan, proinflammatory G protein-coupled receptor that can be activated by medium chain length fatty acids. It is attracting considerable interest as a potential therapeutic target for antagonist ligands in both inflammatory bowel diseases and idiopathic pulmonary fibrosis. Successful screening of more than 300 000 compounds from a small molecule library followed by detailed analysis of some 50 drug-like hits identified 3-((5,6-bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)methyl)-1H-indole as a high affinity and highly selective competitive antagonist of human GPR84. Tritiation of a di-iodinated form of the core structure produced [3H]3-((5,6-diphenyl-1,2,4-triazin-3-yl)methyl)-1H-indole, which allowed effective measurement of receptor levels in both transfected cell lines and lipopolysaccharide-treated THP-1 monocyte/macrophage cells. Although this compound series lacks significant affinity at mouse GPR84, homology modeling and molecular dynamics simulations provided a potential rationale for this difference, and alteration of two residues in mouse GPR84 to the equivalent amino acids in the human orthologue, predicted to open the antagonist binding pocket, validated this model. Sequence alignment of other species orthologues further predicted binding of the compounds as high affinity antagonists at macaque, pig, and dog GPR84 but not at the rat orthologue, and pharmacological experiments confirmed these predictions. These studies provide a new class of GPR84 antagonists that display species selectivity defined via receptor modeling and mutagenesis.

Kallikrein-related peptidase 6 (KLK6) is a secretedserine protease that belongs to the family of tissue kallikreins. Aberrantexpression of KLK6 has been found in different cancers and neurodegenerativediseases, and KLK6 is currently studied as a potential target in these pathologies.We report a novel series of KLK6 inhibitors discovered in a high-throughputscreen within the European Lead Factory program. Structure-guided design basedon docking studies enabled rapid progression of a hit cluster to inhibitorswith improved potency, selectivity and pharmacokinetic properties. Inparticular, inhibitors 32 and 34 have single digit nanomolar potencyagainst KLK6, with over 25-fold and 100-fold selectivity, respectively, againstthe closely related enzyme trypsin. The most potent compound, 32, effectively reduces KLK6-dependentinvasion of HCT116 cells. The high potency in combination with good solubilityand low clearance of 32 make it agood chemical probe for KLK6 target validation in vitro and potentially invivo.

Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.

Cyclic AMP promotes EPAC1 and EPAC2 activation through direct binding to a specific cyclic nucleotide-binding domain (CNBD) within each protein, leading to activation of Rap GTPases, which control multiple cell responses, including cell proliferation, adhesion, morphology, exocytosis, and gene expression. As a result, it has become apparent that directed activation of EPAC1 and EPAC2 with synthetic agonists may also be useful for the future treatment of diabetes and cardiovascular diseases. To identify new EPAC agonists we have developed a fluorescent-based, ultra-high-throughput screening (uHTS) assay that measures the displacement of binding of the fluorescent cAMP analogue, 8-NBD-cAMP to the EPAC1 CNBD. Triage of the output of an approximately 350,000 compound screens using this assay identified a benzofuran oxaloacetic acid EPAC1 binder (SY000) that displayed moderate potency using orthogonal assays (competition binding and microscale thermophoresis). We next generated a limited library of 91 analogues of SY000 and identified SY009, with modifications to the benzofuran ring associated with a 10-fold increase in potency towards EPAC1 over SY000 in binding assays. In vitro EPAC1 activity assays confirmed the agonist potential of these molecules in comparison with the known EPAC1 non-cyclic nucleotide (NCN) partial agonist, I942. Rap1 GTPase activation assays further demonstrated that SY009 selectively activates EPAC1 over EPAC2 in cells. SY009 therefore represents a novel class of NCN EPAC1 activators that selectively activate EPAC1 in cellulae.

The molecular complex between Keap 1 (Kelch-like ECH-associated protein) and Nrf2 (nuclear factor erythroid 2-related factor 2) plays a major role in the regulation of cyto-protective responses to oxidative stress and electrophilic agents. The Keap1-Nrf2 pathway has been established as a therapeutic target for oxidative stress-related conditions including inflammatory, cardiovascular, neurodegenerative diseases and cancer. The European Lead Factory (ELF) is a major European project generating new lead structures for drug discovery programs in the public and private sectors. This is achieved by screening molecular targets against the Joint European Compound Library (JECL) combining compounds contributed by participating pharmaceutical companies or synthesised by chemistry SMEs. Target proposals are submitted to the ELF by European academics and SMEs and selected programs are screened against the library within the European Screening Centre. Hit compounds are provided to the program owner who gains exclusive rights to exploit them for drug discovery or as novel pharmacological tools. The aim of this project was to identify non-electrophilic inhibitors of the Keap1-Nrf2 interaction. Protein-protein interaction (PPI) targets are typically difficult to drug due to large, often quite shallow interaction surfaces between proteins, which along with other factors is associated with PPI screens often returning a high proportion of false positives. Therefore, designing a high-throughput screening (HTS) triage process that confirms target engagement of hits is essential, ideally involving the use of orthogonal biophysical assays. Here we present the successful development of a label-free MicroScale Thermophoresis (MST) assay to identify inhibitors of the Keap1-Nrf2 interaction. 318,132 compounds from the JECL were tested in a fluorescence polarization assay using a fluorescein-labelled Nrf2 peptide mimic. The HTS campaign yielded a hit rate of 0.4% and many false positives compounds were deselected due to fluorescence interference or redox reactivity. Following analytical assessment, visual inspection and legal clearance, 8 compounds were selected for further characterization. Assay development indicated that label-free MST was a suitable platform to confirm target engagement prior to investing in a Chemistry program. Two structurally related hits showed a concentration-dependent binding response to the full-length Keap1 protein in MST, which was abolished after denaturation of the Keap1 protein. These compounds were competitive with one another and with an unlabeled Nrf2 peptide mimic confirming that they were able to disrupt the interaction between Keap1 and Nrf2. Both compounds contain a chiral center and synthesis of the individual enantiomers confirmed that binding was specific to the S-stereoisomer. This data package provided the confidence to initiate a full analogue program with over 110 compounds synthesized and eventually led to ligand-bound crystal structures which helped rationalize the structure-activity relationships. Citation Format: Julie M. Rainard, Angus J. Morrison, Andrew D. Pannifer, Philip S. Jones, Richard J. Mead, Stuart P. McElroy. Characterization of small molecule inhibitors of the Nrf2-Keap1 interaction using MicroScale Thermophoresis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B17.

A major hallmark of Alzheimer's disease (AD) is the formation of neurotoxic aggregates composed of the amyloid-β peptide (Aβ). Aβ has been recognized to interact with numerous proteins, resulting in pathological changes to the metabolism of patients with AD. One such mitochondrial metabolic enzyme is amyloid-binding alcohol dehydrogenase (ABAD), where altered enzyme function caused by the Aβ-ABAD interaction is known to cause mitochondrial distress and cytotoxic effects, providing a feasible therapeutic target for AD drug development. Here we have established a high-throughput screening platform for the identification of modulators to the ABAD enzyme. A pilot screen with a total of 6759 compounds from the NIH Clinical Collections (NCC) and SelleckChem libraries and a selection of compounds from the BioAscent diversity collection have allowed validation and robustness to be optimized. The pilot screen revealed 16 potential inhibitors in the low µM range against ABAD with favorable physicochemical properties for blood-brain barrier penetration.

The mitochondrial enzyme, amyloid binding alcohol dehydrogenase (ABAD), has been shown to mediate the cytotoxic effects of Amyloid-β within the Alzheimer's diseased brain. Mutational studies have shown that ABAD must be catalytically active for cytotoxicity to be observed and therefore the direct inhibition of ABAD may offer a novel therapeutic strategy to treat the disease (1,2,3,4). Our industrial standard high throughput screening (HTS) strategy (5) was accepted into a lucrative pharmaceutical industry backed program that produced a validated hit list of molecules capable of directly inhibiting the ABAD enzyme. To assess the compounds therapeutic potential we are using many different techniques, but our primary screening strategy utilises recombinant ABAD enzyme extract to measure ABAD activity, followed by cell based and biophysical de-selection assays to verify the inhibitory nature, the cell permeability and cytotoxicity of these compounds (see Figure 1). Medicinal chemistry is utilised to optimise the molecules pharmacokinetic properties and potency, whilst x-ray crystallography has been used to visualise the protein-ligand interactions. We have characterised our top 50 hit molecules at both the recombinant protein level (nM potency) and cellular level (low μM potency). Our molecules exhibit favourable chemical characteristics often seen in drug like molecules and several of the most promising molecules have undergone medicinal chemistry to expand on the structural activity relationships (SAR) further.

high-quality starting point can have a significant impact on the outcome of the discovery efforts both in terms of speed and quality.