Identifying ligands for the PHD1 finger of KDM5A through high-throughput screening.

PHD fingers are a type of chromatin reader that primarily recognize chromatin as a function of lysine methylation state. Dysregulated PHD fingers are implicated in various human diseases, including acute myeloid leukemia. Targeting PHD fingers with small molecules is considered challenging as their histone tail binding pockets are often shallow and surface-exposed. The KDM5A PHD1 finger regulates the catalytic activity of KDM5A, an epigenetic enzyme often misregulated in cancers. To identify ligands that disrupt the PHD1-histone peptide interaction, we conducted a high-throughput screen and validated hits by orthogonal methods. We further elucidated structure-activity relationships in two classes of compounds to identify features important for binding. Our investigation offers a starting point for further optimization of small molecule PHD1 ligands.

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments.

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3σ bound to several of its clients, including ERα and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3σ/ERα complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3σ/C-RAF up to 150 µM compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3σ/ERα interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development.

From Tethered to Freestanding Stabilizers of 14-3-3 Protein-Protein Interactions through Fragment Linking.

Small-molecule stabilization of protein-protein interactions (PPIs) is a promising strategy in chemical biology and drug discovery. However, the systematic discovery of PPI stabilizers remains a largely unmet challenge. Herein we report a fragment-linking approach targeting the interface of 14-3-3 and a peptide derived from the estrogen receptor alpha (ERα) protein. Two classes of fragments-a covalent and a noncovalent fragment-were co-crystallized and subsequently linked, resulting in a noncovalent hybrid molecule in which the original fragment interactions were largely conserved. Supported by 20 crystal structures, this initial hybrid molecule was further optimized, resulting in selective, 25-fold stabilization of the 14-3-3/ERα interaction. The high-resolution structures of both the single fragments, their co-crystal structures and those of the linked fragments document a feasible strategy to develop orthosteric PPI stabilizers by linking to an initial tethered fragment.

A Systematic Approach to the Discovery of Protein-Protein Interaction Stabilizers.

Dysregulation of protein-protein interactions (PPIs) commonly leads to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for identifying reversibly covalent small molecules. We explored the scope of disulfide tethering for the discovery of selective PPI stabilizers (molecular glues) using the hub protein 14-3-3σ. We screened complexes of 14-3-3 with 5 biologically and structurally diverse phosphopeptides derived from the 14-3-3 client proteins ERα, FOXO1, C-RAF, USP8, and SOS1. Stabilizing fragments were found for 4/5 client complexes. Structural elucidation of these complexes revealed the ability of some peptides to conformationally adapt to make productive interactions with the tethered fragments. We validated eight fragment stabilizers, six of which showed selectivity for one phosphopeptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious fragment increased 14-3-3σ/C-RAF phosphopeptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3σ provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small-molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic, there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high-resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 153 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated conformational changes within the active site, and key inhibitor motifs that will template future drug development against Mac1.

Iterative computational design and crystallographic screening identifies potent inhibitors targeting the Nsp3 Macrodomain of SARS-CoV-2.

The nonstructural protein 3 (NSP3) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a conserved macrodomain enzyme (Mac1) that is critical for pathogenesis and lethality. While small molecule inhibitors of Mac1 have great therapeutic potential, at the outset of the COVID-19 pandemic there were no well-validated inhibitors for this protein nor, indeed, the macrodomain enzyme family, making this target a pharmacological orphan. Here, we report the structure-based discovery and development of several different chemical scaffolds exhibiting low- to sub-micromolar affinity for Mac1 through iterations of computer-aided design, structural characterization by ultra-high resolution protein crystallography, and binding evaluation. Potent scaffolds were designed with in silico fragment linkage and by ultra-large library docking of over 450 million molecules. Both techniques leverage the computational exploration of tangible chemical space and are applicable to other pharmacological orphans. Overall, 160 ligands in 119 different scaffolds were discovered, and 152 Mac1-ligand complex crystal structures were determined, typically to 1 Å resolution or better. Our analyses discovered selective and cell-permeable molecules, unexpected ligand-mediated protein dynamics within the active site, and key inhibitor motifs that will template future drug development against Mac1.

TPT sulfonate, a single, oral dose schistosomicidal prodrug: In vivo efficacy, disposition and metabolic profiling.

Treatment of schistosomiasis relies precariously on just one drug, praziquantel (PZQ). In the search for alternatives, 15 S-[2-(alkylamino)alkane] thiosulfuric acids were obtained from a previous research program and profiled in mice for efficacy against both mature (>42-day-old) and juvenile (21-day-old) Schistosoma mansoni using a screening dose of 100 mg/kg PO QDx4. One compound, S-[2-(tert-butylamino)-1-phenylethane] thiosulfuric acid (TPT sulfonate), was the most effective by decreasing female and male worm burdens by ≥ 90% and ≥46% (mature), and ≥89% and ≥79% (juvenile), respectively. In contrast, PZQ decreased mature female and male worm burdens by 95% and 94%, respectively, but was ineffective against juvenile stages. Against 7-day-old lung-stage worms, TPT sulfonate was only effective at twice the dose decreasing female and male burdens by 95 and 80%, respectively. Single oral doses at 400 and/or 600 mg/kg across various developmental time-points (1-, 7-, 15-, 21- and/or 42 day-old) were consistent with the QD x4 data; efficacy was strongest once the parasites had completed lung migration, and female and male burdens were decreased by at least 90% and 80%, respectively. In vitro, TPT sulfonate is inactive against the parasite suggesting a pro-drug mechanism of action. In mice, TPT sulfonate is fully absorbed and subject to rapid, non-CYP-mediated, first-pass metabolism that is initiated by desulfation and yields a series of metabolites. The initially-formed free thiol-containing metabolite, termed TP thiol, was chemically synthesized; it dose-dependently decreased S. mansoni and Schistosoma haematobium motility in vitro. Also, when administered as a single 50 mg/kg IP dose, TP thiol decreased 33-day-old S. mansoni female and male burdens by 35% and 44%, with less severe organomegaly. Overall, TPT sulfonate's efficacy profile is competitive with that of PZQ. Also, the characterization of a parasiticidal metabolite facilitates an understanding and improvement of the chemistry, and identification of the mechanism of action and/or target.

Optimization of Phenyl Indole Inhibitors of the AAA+ ATPase p97.

Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.

Allosteric Indole Amide Inhibitors of p97: Identification of a Novel Probe of the Ubiquitin Pathway.

A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts.

2.3 Å resolution cryo-EM structure of human p97 and mechanism of allosteric inhibition.

p97 is a hexameric AAA+ adenosine triphosphatase (ATPase) that is an attractive target for cancer drug development. We report cryo-electron microscopy (cryo-EM) structures for adenosine diphosphate (ADP)-bound, full-length, hexameric wild-type p97 in the presence and absence of an allosteric inhibitor at resolutions of 2.3 and 2.4 angstroms, respectively. We also report cryo-EM structures (at resolutions of ~3.3, 3.2, and 3.3 angstroms, respectively) for three distinct, coexisting functional states of p97 with occupancies of zero, one, or two molecules of adenosine 5'-O-(3-thiotriphosphate) (ATPγS) per protomer. A large corkscrew-like change in molecular architecture, coupled with upward displacement of the N-terminal domain, is observed only when ATPγS is bound to both the D1 and D2 domains of the protomer. These cryo-EM structures establish the sequence of nucleotide-driven structural changes in p97 at atomic resolution. They also enable elucidation of the binding mode of an allosteric small-molecule inhibitor to p97 and illustrate how inhibitor binding at the interface between the D1 and D2 domains prevents propagation of the conformational changes necessary for p97 function.

Structure-Bioactivity Relationship for Benzimidazole Thiophene Inhibitors of Polo-Like Kinase 1 (PLK1), a Potential Drug Target in Schistosoma mansoni.

BACKGROUND: Schistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control. However, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and there is a concern of drug resistance. Thus, there is a need to identify new drugs and drug targets. METHODOLOGY/PRINCIPAL FINDINGS: We show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits a deleterious phenotypic alteration in post-infective larvae (schistosomula or somules). Phenotypic screening and analysis of schistosomula and adult S. mansoni with small molecule inhibitors of huPLK1 identified a number of potent anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor that has completed Phase I clinical trials for treatment of solid tumor malignancies. We then obtained GSKs Published Kinase Inhibitor Sets (PKIS) 1 and 2, and phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a distinctive phenotype distribution. Using principal component analysis (PCA), the phenotypes exhibited by these populations were mapped, visualized and analyzed through projection to a low-dimensional space. The phenotype distribution was found to have a distinct shape and topology, which could be elicited using cluster analysis. A structure-activity relationship (SAR) was identified for the benzimidazole thiophenes that held for both somules and adult parasites. The most potent inhibitors produced marked phenotypic alterations at 1-2 µM within 1 h. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays. CONCLUSIONS/SIGNIFICANCE: The reverse genetic and chemical SAR data support a continued investigation of SmPLK1 as a possible drug target and/or the prosecution of the benzimidazole thiophene chemotype as a source of novel anti-schistosomals.

Structure-Activity Study of Bioisosteric Trifluoromethyl and Pentafluorosulfanyl Indole Inhibitors of the AAA ATPase p97.

Exploratory SAR studies of a new phenyl indole chemotype for p97 inhibition revealed C-5 indole substituent effects in the ADPGlo assay that did not fully correlate with either electronic or steric factors. A focused series of methoxy-, trifluoromethoxy-, methyl-, trifluoromethyl-, pentafluorosulfanyl-, and nitro-analogues was found to exhibit IC50s from low nanomolar to double-digit micromolar. Surprisingly, we found that the trifluoromethoxy-analogue was biochemically a better match of the trifluoromethyl-substituted lead structure than a pentafluorosulfanyl-analogue. Moreover, in spite of their almost equivalent strongly electron-depleting effect on the indole core, pentafluorosulfanyl- and nitro-derivatives were found to exhibit a 430-fold difference in p97 inhibitory activities. Conversely, the electronically divergent C-5 methyl- and nitro-analogues both showed low nanomolar activities.

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

Tetrafluorophenoxymethyl ketone cruzain inhibitors with improved pharmacokinetic properties as therapeutic leads for Chagas' disease.

Inhibition of the cysteine protease cruzain from Trypanosoma cruzi has been studied pre-clinically as a new chemotherapeutic approach to treat Chagas' disease. Efficacious effects of vinylsulfone-based cruzain inhibitors in animal models support this therapeutic hypothesis. More recently, substrate-activity screening was used to identify nonpeptidic tetrafluorophenoxymethyl ketone inhibitors of cruzain that showed promising efficacy in animal models. Herein we report efforts to further optimize the in vitro potency and in vivo pharmacokinetic properties of this new class of cruzain inhibitors. Through modifications of the P1, P2 and/or P3 positions, new analogs have been identified with reduced lipophilicity, enhanced potency, and improved oral exposure and bioavailability.

A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding.

Lead identification to clinical candidate selection: drugs for Chagas disease.

Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing ß-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.

Design and synthesis of hydroxyethylamine (HEA) BACE-1 inhibitors: prime side chromane-containing inhibitors.

The structure activity relationship of the prime region of conformationally restricted hydroxyethylamine (HEA) BACE inhibitors is described. Variation of the P1' region provided selectivity over Cat-D with a series of 2,2-dioxo-isothiochromanes and optimization of the P2' substituent of chromane-HEA(s) with polar substituents provided improvements in the compound's in vitro permeability. Significant potency gains were observed with small aliphatic substituents such as methyl, n-propyl, and cyclopropyl when placed at the C-2 position of the chromane.

Selective and brain-permeable polo-like kinase-2 (Plk-2) inhibitors that reduce α-synuclein phosphorylation in rat brain.

Polo-like kinase-2 (Plk-2) has been implicated as the dominant kinase involved in the phosphorylation of α-synuclein in Lewy bodies, which are one of the hallmarks of Parkinson's disease neuropathology. Potent, selective, brain-penetrant inhibitors of Plk-2 were obtained from a structure-guided drug discovery approach driven by the first reported Plk-2-inhibitor complexes. The best of these compounds showed excellent isoform and kinome-wide selectivity, with physicochemical properties sufficient to interrogate the role of Plk-2 inhibition in vivo. One such compound significantly decreased phosphorylation of α-synuclein in rat brain upon oral administration and represents a useful probe for future studies of this therapeutic avenue toward the potential treatment of Parkinson's disease.

Design and synthesis of highly selective, orally active Polo-like kinase-2 (Plk-2) inhibitors.

Polo-like kinase-2 (Plk-2) is a potential therapeutic target for Parkinson's disease and this Letter describes the SAR of a series of dihydropteridinone based Plk-2 inhibitors. By optimizing both the N-8 substituent and the biaryl region of the inhibitors we obtained single digit nanomolar compounds such as 37 with excellent selectivity for Plk-2 over Plk-1. When dosed orally in rats, compound 37 demonstrated a 41-45% reduction of pS129-α-synuclein levels in the cerebral cortex.

Novel cinnoline-based inhibitors of LRRK2 kinase activity.

Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration.