Discovery and SAR of a novel series of potent, CNS penetrant M4 PAMs based on a non-enolizable ketone core: Challenges in disposition.

This Letter describes the chemical optimization of a novel series of M4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.

Discovery and optimization of a novel series of highly CNS penetrant M4 PAMs based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core.

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp=0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp>10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Selective inhibitor of platelet-activating factor acetylhydrolases 1b2 and 1b3 that impairs cancer cell survival.

Platelet-activating factor acetylhydrolases (PAFAHs) 1b2 and 1b3 are poorly characterized serine hydrolases that form a complex with a noncatalytic protein (1b1) to regulate brain development, spermatogenesis, and cancer pathogenesis. Determining physiological substrates and biochemical functions for the PAFAH1b complex would benefit from selective chemical probes that can perturb its activity in living systems. Here, we report a class of tetrahydropyridine reversible inhibitors of PAFAH1b2/3 discovered using a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) screen of the NIH 300,000+ compound library. The most potent of these agents, P11, exhibited IC50 values of ∼40 and 900 nM for PAFAH1b2 and 1b3, respectively. We confirm selective inhibition of PAFAH1b2/3 in cancer cells by P11 using an ABPP protocol adapted for in situ analysis of reversible inhibitors and show that this compound impairs tumor cell survival, supporting a role for PAFAH1b2/3 in cancer.

Target-based screen against a periplasmic serine protease that regulates intrabacterial pH homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2'-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb's pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.

Characterization of selective exosite-binding inhibitors of matrix metalloproteinase 13 that prevent articular cartilage degradation in vitro.

Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. As a result of high-throughput screening and structure-activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S1' subsite and in an S1/S2* subsite. Type II collagen- and cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Finally, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug-drug interactions in humans.

Development of a highly potent, novel M5 positive allosteric modulator (PAM) demonstrating CNS exposure: 1-((1H-indazol-5-yl)sulfoneyl)-N-ethyl-N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (ML380).

A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).

Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and ß.

Zinc metalloproteinases meprin α and meprin ß are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools, we have developed high throughput screening assays to enable discovery of inhibitors of both meprin α and meprin ß and screened a collection of well characterized pharmaceutical agents (library of pharmaceutically active compounds, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin ß. Kinetic studies revealed competitive (PPNDS) and mixed competitive/noncompetitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin ß active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin ß reported to the date. These results demonstrate the ability of meprin α and ß assays to identify selective compounds and discard artifacts of primary screening.

Discovery, synthesis and characterization of a highly muscarinic acetylcholine receptor (mAChR)-selective M5-orthosteric antagonist, VU0488130 (ML381): a novel molecular probe.

Of the five G-protein-coupled muscarinic acetylcholine receptors (mAChRs; M1-M5), M5 is the least explored and understood due to a lack of mAChR subtype-selective ligands. We recently performed a high-throughput functional screen and identified a number of weak antagonist hits that are selective for the M5 receptor. Here, we report an iterative parallel synthesis and detailed molecular pharmacologic profiling effort that led to the discovery of the first highly selective, central nervous system (CNS)-penetrant M5-orthosteric antagonist, with sub-micromolar potency (hM5 IC50=450 nM, hM5 Ki=340 nM, M1-M4 IC50>30 µM), enantiospecific inhibition, and an acceptable drug metabolism and pharmacokinetics (DMPK) profile for in vitro and electrophysiology studies. This compound will be a powerful tool and molecular probe for the further investigation into the role of M5 in addiction and other diseases.

Transitioning pharmacoperones to therapeutic use: in vivo proof-of-principle and design of high throughput screens.

A pharmacoperone (from "pharmacological chaperone") is a small molecule that enters cells and serves as molecular scaffolding in order to cause otherwise-misfolded mutant proteins to fold and route correctly within the cell. Pharmacoperones have broad therapeutic applicability since a large number of diseases have their genesis in the misfolding of proteins and resultant misrouting within the cell. Misrouting may result in loss-of-function and, potentially, the accumulation of defective mutants in cellular compartments. Most known pharmacoperones were initially derived from receptor antagonist screens and, for this reason, present a complex pharmacology, although these are highly target specific. In this summary, we describe efforts to produce high throughput screens that identify these molecules from chemical libraries as well as a mouse model which provides proof-of-principle for in vivo protein rescue using existing pharmacoperones.

Discovery, design and synthesis of a selective S1P(3) receptor allosteric agonist.

Potent and selective S1P3 receptor (S1P3-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P3-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P3-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P3-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P3-R in a manner that does not disrupt the S1P3-R-S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P3-R allosteric agonists.

Discovery of the first M5-selective and CNS penetrant negative allosteric modulator (NAM) of a muscarinic acetylcholine receptor: (S)-9b-(4-chlorophenyl)-1-(3,4-difluorobenzoyl)-2,3-dihydro-1H-imidazo[2,1-a]isoindol-5(9bH)-one (ML375).

A functional high throughput screen and subsequent multidimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with submicromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1-M4 IC50 > 30 µM), excellent multispecies PK, high CNS penetration, and enantiospecific inhibition.

Discovery of ML326: The first sub-micromolar, selective M5 PAM.

This Letter describes the further chemical optimization of the M5 PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M5 PAMs (M5 EC50 >10µM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M5 PAM, ML326 (VU0467903), (human and rat M5 EC50s of 409nM and 500nM, respectively) with excellent mAChR selectivity (M1-M4 EC50s >30µM) and a robust 20-fold leftward shift of the ACh CRC.

Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes.

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.

A high-throughput assay for the identification of drugs against late-stage Plasmodium falciparum gametocytes.

Recent success in the global reduction campaign against malaria has resulted in the possibility that it may be feasible to drastically reduce or even eradicate malaria even without the introduction of a vaccine. However, while there has been significant effort to design the next generation of antimalarial drugs, one area that is underrepresented in the current antimalarial pharmacopeia is that of transmission blocking drugs directed at late-stage gametocytes. Here we describe the development of a robust and simple assay that is amenable to a high throughput format for the discovery of new antigametocyte drugs.

A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors.

Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL and in eventually understanding the link between PREPL and HCS.

Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesterase-1 inhibitors.

National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-ß-lactams (ABLs), as potent (IC(50) values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

NH-1,2,3-Triazole-based Inhibitors of the VIM-2 Metallo-ß-Lactamase: Synthesis and Structure-Activity Studies.

Metallo-ß-lactamases (MBL) are an emerging cause of bacterial resistance to antibiotic treatment. The VIM-2 ß-lactamase is the most commonly encountered MBL in clinical isolates worldwide. Described here are potent and selective small molecule inhibitors of VIM-2 containing the arylsulfonyl-NH-1,2,3-triazole chemotype that potentiate the efficacy of the ß-lactam, imipenem, in E. coli.

Oxime esters as selective, covalent inhibitors of the serine hydrolase retinoblastoma-binding protein 9 (RBBP9).

We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying enzyme's active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases.

Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput screening.

The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide) (BIIE0246). Presented here are five potent, selective, and publicly available Y2R antagonists identified by a high-throughput screening approach. These compounds belong to four chemical scaffolds that are structurally distinct from the peptidomimetic BIIE0246. In functional assays, IC(50) values between 199 and 4400 nM against the Y2R were measured, with no appreciable activity against the related NPY-Y1 receptor (Y1R). Compounds also displaced radiolabeled peptide YY from the Y2R with high affinity (K(i) values between 1.55 and 60 nM) while not displacing the same ligand from the Y1R. In contrast to BIIE0246, Schild analysis with NPY suggests that two of the five compounds behave as competitive antagonists. Profiling against a panel of 40 receptors, ion channels, and transporters found in the central nervous system showed that the five Y2R antagonists demonstrate greater selectivity than BIIE0246. Furthermore, the ability of these antagonists to penetrate the blood-brain barrier makes them better suited for pharmacological studies of Y2R function in both the brain and periphery.