[Show abstract][Hide abstract] ABSTRACT: Evidence from preclinical and human studies demonstrate that arginine vasopressin and vasopressin receptor 1a (V1a-R) play a crucial role in the pathophysiology of psychiatric disorders such as anxiety, depression and post-traumatic stress disorder (PTSD). The lack of selective, brain-penetrant and orally active V1a receptor antagonists has hampered the progress in our understanding of the precise/complete pharmacological role of V1a-R in central nervous system disorders and the development of novel treatments for these disorders. The optimization of promising V1a-R antagonist lead compounds and testing in known and relevant in vivo animal models will be a significant step forward in our understanding of the role of V1a-R in CNS disorders and in the development of novel treatments of anxiety, depression and PTSD. The lack of published data regarding brain-penetration and off-target selectivity, along with intellectual property barriers of the known V1a-R antagonists precludes their potential usefulness as CNS agents. As a result, there is a substantial need to discover centrally active V1a-R antagonists that are useful as in vivo tools to better understand the pharmacological role of V1a-R in CNS disorders with the potential as clinically therapeutic agents. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports ML389 as a highly potent V1a-R antagonist with an IC50 of 40 nM, and high selectivity (selectivities vs. vasopressin 1b receptor, vasopressin 2 receptor, and the oxytocin receptor of >1250 fold). ML389 was identified by medicinal chemistry optimization of lead compounds. A set of pharmacokinetic analyses show that ML389 has good brain penetration, no significant activity at four human cytochrome P450 subtypes, high binding for human and rodent plasma protein, and an encouraging in vivo pharmacokinetic profile in mice. Broad selectivity screening data against a panel of receptors, transporters, and ion channels suggest that ML389 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. ML389 serves as a novel V1a-R antagonist that can be developed as a therapeutic for the treatment of psychiatric disorders such as anxiety, depression and PTSD.
[Show abstract][Hide abstract] ABSTRACT: Lipoprotein-associated phospholipase A(2) (Lp-PLA(2) or PLA(2)G7) binds to low-density lipoprotein (LDL) particles, where it is thought to hydrolyze oxidatively truncated phospholipids. Lp-PLA(2) has also been implicated as a pro-tumorigenic enzyme in human prostate cancer. Several inhibitors of Lp-PLA(2) have been described, including darapladib, which is currently in phase 3 clinical development for the treatment of atherosclerosis. The selectivity that darapladib and other Lp-PLA(2) inhibitors display across the larger serine hydrolase family has not, however, been reported. Here, we describe the use of both general and tailored activity-based probes for profiling Lp-PLA(2) and inhibitors of this enzyme in native biological systems. We show that both darapladib and a novel class of structurally distinct carbamate inhibitors inactivate Lp-PLA(2) in mouse tissues and human cell lines with high selectivity. Our findings thus identify both inhibitors and chemoproteomic probes that are suitable for investigating Lp-PLA(2) function in biological systems.
No preview · Article · Dec 2012 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jun 2012 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: FTY720 (fingolimod, Gilenya ®) is a sphingosine analog prodrug that induces lymphocyte sequestration in secondary lymphoid organs, resulting in peripheral lymphopenia. Lymphopenia by FTY720 is characterized by a dosedependent and sustained reduction of circulating T and B lymphocytes within hours of administration, accounting for a 70% reduction of CD4 + T cells, 90% reduction of CD8 + T-cells, and >50% reduction of CD19 + B-cells, with a single administration. Mechanistically, lymphocyte sequestration following FTY720 administration in vivo is due to blockade of lymphocyte egress into efferent lymph. The induction of lymphopenia by FTY720 requires the generation of its active phosphate intermediate, FTY720-P, a step catalyzed by phosphorylation of FTY720 by the ubiquitous sphingosine-kinase 2. In turn, FTY720-P becomes a potent subnanomolar agonist on four of the five sphingosine-1 phosphate (S1P) receptors (S1P 1,3,4,5). Clinically, FTY720 has recently become the first oral therapy to be approved for the treatment of relapsingremitting multiple sclerosis. As a research tool, FTY720 has been critical in providing mechanistic insights into lymphocyte trafficking in physiology and disease. Although FTY720-P displays similar potency across S1P receptors, it is the S1P 1 subtype, a class-A-type G i-coupled GPCR, that is the sole mediator of FTY720-P's ability to induce lymphopenia. S1P 1 is broadly expressed in mammals, with relatively high expression found in neuronal, immune and endothelial cells, and has proven to be critical for vascular development in rodents. The aim of this review is to highlight FTY720's efficacy as an immunosuppressant, examine its mechanisms of action, and the emergence of mono-selective S1P 1 agonist therapies for autoimmune diseases.
No preview · Article · May 2012 · Current Immunology Reviews
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the cytosolic enzyme type II platelet-activating factor acetylhydrolase (PAFAH2) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. In support of this theory, over-expression of PAFAH2 has been shown to reduce oxidative stress-induced cell death . However, no selective inhibitors of PAFAH2 are known for investigation of PAFAH2 biology. We initiated a fluorescence polarization activity-based protein profiling (FluoPol-ABPP) HTS campaign to identify potential inhibitors of PAFAH2 (AIDs 492956 and 493030). The assay also served as a counterscreen for inhibitor discovery for the related enzyme, plasma PAFAH (pPAFAH; AIDs 463082, 463230). Interestingly, the triazole urea SID 7974398—a top lead in the lysophospholipase (LYPLA1) inhibitor screen from which we derived a dual inhibitor of LYPLA1/LYPLA2 (ML211) and inhibitor of ABHD11 (ML226)—was also a top hit in the PAFAH2 HTS assay. Given that triazole ureas were previously found to have tunable potency and selectivity, low cytotoxicity, and good activity in situ, we endeavored to derive a PAFAH2-selective probe from the triazole urea scaffold. The medchem optimized probe (ML225, SID 103913572) is highly potent against its target enzyme (IC50 = 3 nM), and is active in situ at sub-nanomolar concentrations. ML225 is at least 333-fold selective for all other serine hydrolases (~20) assessed by gel-based competitive activity-based protein profiling, and is selective for other PAFAH enzymes. ML225 inhibits PAFAH2 by carbamoylating the active site serine. The complete properties, characterization, and synthesis of ML225 are detailed in this Probe Report.
[Show abstract][Hide abstract] ABSTRACT: Malaria is one of the most prevalent human parasitic diseases and is a global health issue accounting for >600,000 deaths annually. For survival, the Plasmodium falciparum (Pf) malaria parasite requires the action of a number of metallo-aminopeptidases that each display restricted amino acid specificities, including PfM1MAA (membrane alanine aminopeptidase), PfM17LAP (leucine aminopeptidase), and PfM18AAP (aspartyl aminopeptidase), which are thought to act in concert to degrade host erythrocyte hemoglobin that the parasite uses as a source of amino acid building blocks for the synthesis of its own proteins. Since there are several small molecule inhibitors of PfM1MAA and PfM17LAP, and very few small molecule inhibitors of PfM18AAP, we set out to identify small molecule inhibitors of PfM18AAP. Biochemical assays employing rPfM18AAP, native PfM18AAP, recombinant Fasciola hepatica cathepsin L1 (rFhCTSL1), rPfM1MAA, rPfM17LAP, and rhM18, and cell-based parasite growth inhibition and cytotoxicity assays were used to identify CID23724194 (from the NIH MLSMR) as a viable starting point for medicinal chemistry optimization. Two rounds of structure-activity relationship studies were performed to generate the probe ML369 (CID56846691). The probe is the best-in-class small molecule inhibitor of PfM18AAP; however, certain liabilities discussed in detail in the probe report limit its usefulness. When the probe is used as recommended, the probe is “fit-for-purpose” and should be useful for advancing the field.
[Show abstract][Hide abstract] ABSTRACT: Endocannabinoids (ECs) are a unique group of lipids that function as chemical messengers in the nervous system. The two principle ECs thus far identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction. Because ECs are synthesized and released on demand and then rapidly degraded to terminate signaling, the metabolic pathways that govern EC turnover directly influence the magnitude and duration of neuronal signaling events. Three of the key enzymes responsible for 2-AG hydrolysis (and thus inactivation) are monoacylglycerol lipase (MAGL), abhydrolase domain containing proteins 6 (ABHD6), and ABHD12. In an effort to develop more potent, in vivo active ABHD6 inhibitors, both to serve as control probes for the recently developed dual DAGL-β/ABHD6 inhibitor ML294 and for biological investigation of ABHD6 in its own right, we initiated a competitive activity-based protein profiling (ABPP)-guided medicinal chemistry campaign to identify and optimize ABHD6 inhibitors based on the triazole urea scaffold. This campaign, made possible by previous MLPCN probe development efforts for LYPLA1/2 (ML211), PAFAH2 (ML225), ABHD11 (ML226), and DAGL-β (ML294) yielded the medchem-optimized probes ML295 (SID 125269138) and ML296 (SID 125269096). These compounds exhibit low nM potency in vitro and in situ, high selectivity among the serine hydrolase superfamily, and strong inhibitory activity in vivo. The key distinguishing feature of ML295 compared to ML296 is that the former compound is brain-penetrant, while the latter compound is peripherally-restricted in mice. These compounds, together, thus provide key pharmacological tools for dissecting the functions of ABHD6 in central vrs peripheral tissues. These successful probe development efforts further confirm the privileged nature of the triazole urea scaffold for potent and selective inhibition of serine hydrolase targets in living systems.
[Show abstract][Hide abstract] ABSTRACT: The Scripps Research Institute Molecular Screening Center (SRIMSC) recently completed a fluorescence-polarization activity-based protein profiling (fluopol-ABPP) high throughput screening (HTS) campaign to identify inhibitors of protein phosphatase methylesterase-1 (PME-1). This campaign unveiled a phenomenal class of potent and selective inhibitors, the aza-beta lactams (ABLs), one of which, ML174, showed exceptional in situ and in vivo potency and selectivity for PME-1. During medicinal chemistry investigation of the ABLs for PME-1, we observed that one of the common anti-targets was the uncharacterized serine hydrolase abhydrolase domain containing protein 10 (ABHD10). This fortuitous discovery of inhibitor leads was of particular interest to us, as we had recently uncovered some exciting evidence that ABHD10 functions as a lipase in situ. A principle goal of post-genomic research is to elucidate the molecular and cellular roles of uncharacterized enzymes like ABHD10, an investigation which profits significantly from chemical tools for precise regulation of enzyme activity. Given that no selective inhibitors of ABHD10 have yet been reported in the literature, we completed a medicinal chemistry campaign to optimize an ABL probe for ABHD10, which is presented herein as ML257. This probe is highly potent against ABHD10 in vitro (IC50 = 17 nM), in situ (IC50 = 28 nM), and in vivo (active at 25 mg/kg, i.p., in mice), and exhibits remarkable selectivity among 40+ other serine hydrolases. Importantly, this probe demonstrates the potential for exploiting fortuitous inhibitor leads for orthogonal, “anti-target” enzymes, thus maximizing the benefits of a single HTS campaign, and highlights the “privileged” nature of the ABL scaffold for serine hydrolase inhibitor development.
[Show abstract][Hide abstract] ABSTRACT: The opioid receptors are a subfamily of the family A G protein-coupled opioid receptor superfamily and consist of mu (OPRM1), delta (OPRD1), and kappa (OPRK1), all of which activate inhibitory G proteins. The dynorphins act as endogenous agonists of OPRK1 to activate a variety of signaling transduction pathways including those involving mitogen activated protein kinases (MAPK). Activation of OPRK1 leads to a number of physiological effects implicating a role for these receptors in addiction, dysphoria and reward. Hence, OPRK1 antagonists are being explored for their effects in the treatment of cocaine addiction, depression, and feeding behavior and have been proposed as a treatment for psychosis and schizophrenia. While a number of OPRK1 antagonists have been identified, all of the prototypic antagonists are very long-acting, exhibit unusual pharmacology, exhibit delayed onset of action, and are associated with serious safety concerns. Very few drug-like OPRK1 antagonists have been developed. New OPRK1 antagonists possessing novel scaffolds and improved selectivity are needed as pharmacological tools to better understand the OPRK1-dynorphin system and as potential pharmacotherapies. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports ML350 as a highly potent OPRK1 antagonist with an IC50 of 9–16 nM, with high selectivity (selectivities vs. OPRD1 and OPRM1 of 219–382–fold and 20–35–fold, respectively). ML350 was identified by high-throughput screening using a cell-based Tango™-format assay. A set of pharmacokinetic analyses show that ML350 has high passive membrane permeability, good brain penetration, no significant activity at three of four human cytochrome P450 subtypes, high binding for rodent plasma protein and modest binding for human plasma protein, and an encouraging in vivo pharmacokinetic profile in rats. ML350 was submitted to CEREP for broad panel screening against a panel of receptors, transporters, and ion channels; the data suggest that ML350 is generally inactive against a broad array of off targets and does not likely exert unwanted effects. Importantly, ML350 was shown to have a reversible analgesic effect when challenged with an OPRK1 agonist in a tail flick assay in mice. ML350 serves as a novel OPRK1 antagonist that can be developed as a therapeutic for the treatment of a variety of disorders involving the OPRK1-dynorphin system.
[Show abstract][Hide abstract] ABSTRACT: Glutathione transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Using activity-based proteomic methods, glutathione S-tranferase omega (GSTO1) has been shown to be overexpressed in human cancer cell lines that show enhanced aggressiveness. Other studies have implicated GSTO1 in chemotherapeutic resistance. Cancer remains one of the most life-threatening diseases for which effective treatments and cures are lacking. Recently, targeted therapeutics (i.e., selective inhibitors that block individual enzymes) have shown great promise for the treatment of cancer. Inhibiting GSTO1 may thus offer a new therapeutic strategy for cancer. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective GSTO1 inhibitor probe, ML175, by high-throughput screening using fluorescence polarization-activity-based protein profiling (FluoPol-ABPP), and several rounds of gel-based competitive activity-based protein profiling (ABPP) secondary assays employing SAR analysis of selected compounds. ML175, a hindered alpha-chloroacetamide, was identified as a highly potent and selective inhibitor of the target enzyme GSTO1. ML175 has an IC50 of 28 nM, greater than 350-fold selectivity against potential anti-targets as assessed by competitive ABPP profiling, and has been shown to be active in situ, completely inhibiting GSTO1 at 250 nM compound concentration. As determined from LC-MS/MS analysis, ML175 is an activity-based inhibitor; it covalently labels the active site cysteine nucleophile of GSTO1. ML175 has favorable stability, solubility, and cytotoxicity profile, and improves on the current state of the art for GSTO1 inhibitors. Taken together, our findings suggest that it is very possible to develop potent and selective probes based on tempered electrophilic scaffolds, and that ML175 will be a highly successful probe for biochemical investigation of GSTO1.
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress has been implicated as an underlying inflammatory factor in several disease pathologies, including cancer, atherosclerosis, aging, and various neurodegenerative disorders. Phospholipids in particular are susceptible to oxidative damage, and it is thought that the plasma platelet-activating factor acetylhydrolase (pPAFAH, a.k.a. PLA2G7) may facilitate turnover of oxidized phospholipids via hydrolysis of their oxidatively truncated acyl chains. However, there are no commercially available, selective, and in vivo-active inhibitors for investigation of pPAFAH biology. As such, we initiated a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high throughput screening (HTS) campaign to identify potential inhibitors of pPAFAH and three PAFAH family members: PAFAH2, PAFAHb2, and PAFAH1b3. The pPAFAH HTS campaign revealed a large number (approximately 5000) of potential lead compounds, but secondary gel-based screening of approximately 150 cherry picked top inhibitors quickly revealed the carbamate as a promising scaffold for inhibitor design. Given that carbamate inhibitors for serine hydrolase enzymes have previously been found to have tunable potency and selectivity and good activity in vivo, we endeavored to derive a pPAFAH-selective probe from the carbamate scaffold. In this effort, we were aided by the recent identification of a lead pPAFAH carbamate inhibitor from a small in-house library. By combining elements of both the HTS carbamate hits and our in-house lead, the medchem optimized probe (ML256, SID 125269079) is highly potent against its target enzyme (IC50 = 31 nM mouse isoform; 6 nM human isoform), and is active in situ and in vivo, showing excellent oral bioavailability and blood-brain barrier penetration. ML256 is at least 322-fold selective for all other brain serine hydrolases (approximately 20) assessed by gel-based competitive activity-based protein profiling (ABPP), and is selective for other PAFAH enzymes as determined by both gel-based screening and Multidimensional Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis (ABPP-MudPIT). ML256 inhibits pPAFAH by irreversible carbamoylation of the active site serine.
[Show abstract][Hide abstract] ABSTRACT: Heterotrimeric G-protein coupled receptors (GPCRs), the largest family of membrane-bound receptors, are major targets for therapeutic applications due to their broad tissue distribution, structural diversity, varied modes of action, and disease-associated mutations. The recently de-orphanized GPCR GPR7 is distributed predominantly in the central nervous system. Neuropeptides W (NPW) and B (NPB) have been identified as endogenous ligands of GPR7. GPR7 represents a new and expanding target for drug development as it has been demonstrated to modulate the release of pituitary-derived hormones and implicated in feeding behavior, the development of obesity, and mediating the inflammatory pain response. There is a significant medical need for novel compounds for treating chronic pain that are effective, long lasting, and safe. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a potent and selective GPR7 antagonist probe, ML250, by high-throughput screening using a cell-based fluorescence assay. ML250 inhibits human GPR7 expression in the presence of a GPR7 agonist NPW with an IC50 of 124nM–244nM. In a counterscreen for melanin-concentrating hormone receptor 1 (MCH1) antagonism in the presence of an agonist, ML250 has an IC50 of >20 μM, resulting in a selectivity for GPR7 of 82 – >161 fold. ML250 is nontoxic to U2OS cells, with a CC50 of > 20 μM. We previously reported ML181, a 272 nM antagonist probe that is 14-fold selective for GPR7 over MCH1, and the first reported small molecule modulator of GPR7. ML250 has comparable potency but greater selectivity than ML181 and will be a valuable tool in elucidating the diverse roles of this receptor in physiological and pathological processes. ML250 also represents a potential therapeutic option in treating chronic pain.
[Show abstract][Hide abstract] ABSTRACT: Several nuclear receptors (NRs) are still characterized as orphan receptors because endogenous ligands have not yet been identified for these proteins. Evidence is growing suggesting the retinoic acid receptor-related orphan receptors (RORs) bind to, and are modulated by oxysterols. Recently, we discovered that the synthetic LXRα agonist T0901317 (ML125) was a potent inverse agonist of RORα and RORγ. Structure activity relationship (SAR) studies quickly revealed a strategy to remove the LXRα activity from the ML125 chemical scaffold which led to ML124. ML124 represented the first synthetic RORα/RORγ dual inverse agonist devoid of LXRα activity. While there appear to be clear non-overlapping roles for RORα and RORγ, chemical probes that are isoform selective are needed to dissect this biology. We described the identification of a selective RORα synthetic ligand, ML176, which directly binds to RORα, but not other RORs, and functions as a selective inverse agonist of RORα in cell-based assays. Here we describe the identification of a selective RORγ synthetic ligand, ML310, which functions as an inverse agonist. We show that ML310 can displace T1317 in a binding assay and does interact with RORγ protein to stabilize the protein in hydrogen-deuterium exchange (HDX)-based experiments. In cotransfection assays, ML310 suppresses transcription activity in both GAL4-RORγ ligand binding domain (LBD) and full-length RORγ contexts. Furthermore, treatment of EL-4 cells with ML310 results in suppression of gene expression and production of IL-17. These data strongly suggest that ML310 is a potent and efficacious RORγ modulator and represses its activity. Thus, we have identified the first synthetic RORγ selective inverse agonist, and this compound can be utilized as a chemical tool to probe the function of this receptor both in vitro and in vivo. Additionally, our data suggests that RORγ inverse agonists may hold utility for the treatment of autoimmune disorders.
[Show abstract][Hide abstract] ABSTRACT: The Hepatitis C virus (HCV) is a major cause of liver failure and hepatocellular carcinoma, with about 170 million people infected worldwide. Replication of HCV in human cells requires the action of the HCV non-structural protein 3 (NS3), which exhibits both protease and helicase activities. Since there are numerous NS3 protease inhibitors but few NS3 helicase inhibitors, the goal here was to develop specific NS3 helicase inhibitors. During assay development, we discovered that trace components in the dye mixtures thioflavine S and primuline were potent inhibitors of the NS3 helicase. Based on the common structure of these components we designed ML283. The probe candidate is a potent inhibitor of the NS3 helicase enzyme and can be synthesized in the necessary quantities for further investigation. ML283 is more potent and specific than other recently reported NS3 helicase inhibitors under the same assay conditions, allowing a direct comparison. Preliminary experiments indicate that this compound is able to penetrate cells and inhibit HCV replication with no significant cytotoxicity. Thus the probe would be a useful tool for the investigation of viral helicases.
[Show abstract][Hide abstract] ABSTRACT: Protein homeostasis, also called proteostasis, is critical for cellular health and its dysregulation is implicated in aging, cancer, metabolic disease, and neurodegenerative disorders. Proteostasis involves compartmentalized cellular responses (e.g. Heat Shock Response in the cytoplasm, Unfolded Protein Response in the mitochondria and endoplasmic reticulum) that limit protein misfolding and aggregation. Diseases of protein conformation are characterized by inefficient induction of these responses. As a result, identification of molecules that activate cellular stress responses and increase proteostasis may be useful for maintaining cell health. Here, we report on high throughput screening efforts that resulted in identification of a novel activator of heat shock protein 70 (Hsp70): ML346. Probe ML346 belongs to the barbituric acid scaffold. ML346 induces HSF-1-dependent chaperone expression and restores protein folding in conformational disease models. These effects are mediated by novel mechanisms involving FOXO, HSF-1, and Nfr-2.
[Show abstract][Hide abstract] ABSTRACT: The protein arginine deiminases (PADs) are a family of Ca(2+)-dependent enzymes that catalyze the conversion of peptidyl-arginine to peptidyl-citrulline in numerous protein substrates. Disruption of normal PAD activity plays a role in the pathogenesis of multiple inflammatory diseases such as rheumatoid arthritis (RA), chronic obstructive pulmonary disease, ulcerative colitis, multiple sclerosis, psoriasis, Alzheimer’s disease, and in various cancers. PAD inhibitors described in the literature have been useful chemical tools to study the role of PAD enzymes in inflammatory diseases and cancer biology. Most published PAD inhibitors are mechanism-based inactivators belonging to the halogen-amidine chemotype. For emerging targets such as the PADs, it can be difficult to distinguish compound-specific effects from those truly resulting from enzyme inhibition. We therefore initiated a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high throughput screening (HTS) campaign to identify a second PAD inhibitor chemotype. The PAD4 HTS campaign identified the natural product streptonigrin (SID 11532976) as an irreversible PAD4-specific inhibitor. We describe herein the medicinal chemistry optimization of streptonigrin to the pan PAD probe ML325 (SID 118043677). ML325 inhibits PAD1, 2, 3, and 4 in vitro with IC50 values of 70 nM, 200 nM 170 nM, and 240 nM respectively. In a kinetic assay of inhibition more appropriate for irreversible inhibitors, ML325 has kinact/KI values of 3500, 7300, 1900, and 5300 M(−1)min(−1) for PAD1, 2, 3 and 4 respectively; indicating it has less than 4-fold selectivity among the four family members. Despite its promiscuity within the PAD family, ML325 exhibits high selectivity vs. more than 20 cysteine-reactive proteins as assayed by activity-based protein profiling. ML325 was also demonstrated to inhibit all four PAD isozymes irreversibly and to be non-cytotoxic to NIH-3T3 cells. The complete properties, characterization, and synthesis of ML325 are detailed in this report.
[Show abstract][Hide abstract] ABSTRACT: Malaria is one of the most prevalent human parasitic diseases and is a global health issue accounting for >600,000 deaths annually. For survival, the Plasmodium falciparum (Pf) malaria parasite requires the action of a number of metallo-aminopeptidases, including PfM1MAA (membrane alanine aminopeptidase), PfM17LAP (leucine aminopeptidase), and PfM18AAP (aspartyl aminopeptidase). Each enzyme displays restricted amino acid specificity, and they are thought to act in concert to degrade proteins (i.e., host erythrocyte hemoglobin) that the parasite uses to generate a pool of amino acids which are employed as building blocks for the synthesis of its own proteins. Since there were very few small-molecule inhibitors of PfM17LAP and no selective inhibitors relative to PfM1AAP (or PfM18AAP), we set out to identify new potent and selective small-molecule inhibitors of this enzyme. Biochemical assays employing enzymatically active recombinant PfM17LAP (rPfM17LAP), as well as recombinant Fasciola hepatica cathepsin L1 (rFhCTSL1), rPfM1MAA, rPfM18AAP, and human M17LAP (rhuM17), and cell-based parasite growth inhibition and cytotoxicity assays were used to identify CID 2466 (from the NIH MLSMR) as a viable starting point for SAR analysis. Three rounds of structure-activity relationship studies were performed to generate a panel of probe candidate compounds. Ultimately, the compound hit, CID 2466, also known as bufexamac, was nominated as the probe ML392. When the probe and analogues are used as recommended, they are “fit-for-purpose” and should be useful for advancing the search for new antimalarial drugs directed at PfM17LAP.
[Show abstract][Hide abstract] ABSTRACT: Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of regulatory proteins that play key roles in cell growth and signaling. Multiple oncogenes, including HRAS and SRC, require palmitoylation for malignant transformation. Lysophospholipase 1 (LYPLA1) has been identified as a candidate protein palmitoyl thioesterase responsible for HRAS depalmitoylation in mammalian cells. Seeking chemical tools to investigate LYPLA biochemical pathway involvement and potential roles in cancer pathogenesis, we conducted a fluorescence polarization-based competitive activity-based protein profiling (fluopol-ABPP) HTS campaign to identify inhibitors of LYPLA1 and the structurally related LYPLA2. HTS identified a lead triazole urea micromolar inhibitor, which we optimized as dual LYPLA1/LYPLA2 inhibitor ML211, and reversible compounds ML348 and ML349 that act as selective LYPLA1 and LYPLA2 inhibitors, respectively. These compounds represented an important advance in chemical tools for investigating the biological function(s) of LYPLA1 and LYPLA2. However, ML211, owing to its modest solubility, does not have demonstrated in vivo activity. With the goal of developing an irreversible, in vivo-active dual LYPLA1/LYPLA2 inhibitor, we investigated SAR around a carbamate scaffold, from which we optimized ML378, a dual LYPLA1/LYPLA2 inhibitor with nM potency and demonstrated peripheral in vivo activity.
[Show abstract][Hide abstract] ABSTRACT: Opiates such as morphine are the choice analgesic in the treatment of chronic pain due to their potent and rapid action. Opioid receptors belong to the family of G protein-coupled receptors (GPCRs), one of the largest gene families in the mammalian genome. The OPRM1 gene encodes the mu opioid receptor, which is the primary site of action for morphine and other commonly used opioids such as heroin, fentanyl, and methadone. The long-term use of opiates is limited because of the development of tolerance and dependence, as well as respiratory suppression and constipation. Due to their clinical importance, various strategies have been considered for making opiates more effective while curbing liabilities such as addiction. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, delta opioid receptor (OPRD1) ligands have been useful in enhancing morphine’s potency, but the underlying molecular basis is not understood. It has been shown that modulation of receptor function by physical association between OPRM1 and OPRD1 is a potential mechanism; heteromerization of OPRM1 with OPRD1 leads to the modulation of receptor binding and signaling properties. It has further been shown that the selective activation of the OPRM1-OPRD1 heteromer by a combination of OPRM1 agonist with OPRD1 antagonist can be blocked by antibodies that selectively recognize the heteromer. Therefore, the identification of compounds that selectively activate OPRM1-OPRD1 heterodimerization may have potential in the treatment of pain and alleviate unwanted effects associated with opiate use. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), reports here an agonist for OPRM1-OPRD1 heterdimerization, ML335, with an EC50 of 403 nM, and selectivities vs. OPRM1, OPRD1, and HTR5A of 37, 2.7, and >99, respectively.
[Show abstract][Hide abstract] ABSTRACT: Endocannabinoids (ECs) are a unique group of lipids that function as chemical messengers in the nervous system. The two principle ECs thus far identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). These compounds have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction. Because ECs are synthesized and released on demand and then rapidly degraded to terminate signaling, the metabolic pathways that govern EC turnover directly influence the magnitude and duration of neuronal signaling events. There is strong evidence that two serine hydrolases, diacylglycerol lipase-alpha and -beta (DAGL-α and -β) function as 2-AG synthetic enzymes both in vitro and in vivo. However, because constitutive gene disruption, the only currently available means to investigate DAGL-α/β biology due to a lack of selective chemical inhibitors, can result in compensatory effects and network-wide changes, there is still uncertainty surrounding the extent to which DAGL-α/β contribute to 2-AG-mediated signaling. In an effort to provide chemical tools for manipulation of DAGL-β activity, we initiated a competitive activity-based protein profiling (ABPP) screen of triazole urea compounds to identify selective enzyme inhibitors. This campaign, made possible by previous inhibitor development efforts for LYPLA1/2 (ML211), PAFAH2 (ML225), and ABHD11 (ML226) based on the triazole urea scaffold, yielded the medchem optimized probe ML294 (SID 125269120). ML294 is highly potent against its target enzyme (IC50 = 56 nM in vitro; 12 nM in situ), and is active in vivo, showing both oral bioavailability and blood-brain barrier penetration. Out of more than 20 serine hydrolases (SHs) profiled by gel-based competitive ABPP, ML294 is observed to have one anti-target, alpha/beta hydrolase domain-containing protein 6 (ABHD6). Otherwise, ML294 is at least 35-fold selective for all other brain SHs (approximately 20) assessed by gel-based competitive ABPP and 7-fold selective vs. its closest homolog, DAGL-α. To control for ABHD6-directed activity in biological studies, we also developed a structurally related ABHD6-selective control “anti-probe”, ML295, also based on the triazole urea scaffold. The complete properties, characterization, and synthesis of ML294 are detailed in this report, and full details of ABHD6 inhibitors are detailed in the Probe Report for ML295 and ML296.