Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/ß-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors.

Discovery of the Potent and Selective ATR Inhibitor Camonsertib (RP-3500).

ATR is a key kinase in the DNA-damage response (DDR) that is synthetic lethal with several other DDR proteins, making it an attractive target for the treatment of genetically selected solid tumors. Herein we describe the discovery of a novel ATR inhibitor guided by a pharmacophore model to position a key hydrogen bond. Optimization was driven by potency and selectivity over the related kinase mTOR, resulting in the identification of camonsertib (RP-3500) with high potency and excellent ADME properties. Preclinical evaluation focused on the impact of camonsertib on myelosuppression, and an exploration of intermittent dosing schedules to allow recovery of the erythroid compartment and mitigate anemia. Camonsertib is currently undergoing clinical evaluation both as a single agent and in combination with talazoparib, olaparib, niraparib, lunresertib, or gemcitabine (NCT04497116, NCT04972110, NCT04855656). A preliminary recommended phase 2 dose for monotherapy was identified as 160 mg QD given 3 days/week.

Broadening the spectrum of ß-lactam antibiotics through inhibition of signal peptidase type I.

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

RP-3500: A Novel, Potent, and Selective ATR Inhibitor that is Effective in Preclinical Models as a Monotherapy and in Combination with PARP Inhibitors.

Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.

Conversion of systemically-distributed triazole-based stearoyl-CoA desaturase (SCD) uHTS hits into liver-targeted SCD inhibitors.

It has been demonstrated that once-a-day dosing of systemically-distributed SCD inhibitors leads to adverse events in eye and skin. Herein, we describe our efforts to convert a novel class of systemically-distributed potent triazole-based uHTS hits into liver-targeted SCD inhibitors as a means to circumvent chronic toxicity.

Biological activity and preclinical efficacy of azetidinyl pyridazines as potent systemically-distributed stearoyl-CoA desaturase inhibitors.

Potent and orally bioavailable SCD inhibitors built on an azetidinyl pyridazine scaffold were identified. In a one-month gDIO mouse model of obesity, we demonstrated that there was no therapeutic index even at low doses; efficacy in preventing weight gain tracked closely with skin and eye adverse events. This was attributed to the local SCD inhibition in these tissues as a consequence of the broad tissue distribution observed in mice for this class of compounds. The search for new structural scaffolds which may display a different tissue distribution was initiated. In preparation for an HTS campaign, a radiolabeled azetidinyl pyridazine displaying low non-specific binding in the scintillation proximity assay was prepared.

2-Aryl benzimidazoles: human SCD1-specific stearoyl coenzyme-A desaturase inhibitors.

A series of potent, benzimidazole-based SCD inhibitors which demonstrate selectivity for the hSCD1 enzyme over the hSCD5 isoform are described. The compounds possess suitable cellular activity and pharmacokinetic properties which render them capable of inhibiting liver SCD activity in a mouse pharmacodynamic assay. These 2-aryl benzimidazoles may serve as valuable tools for studying selective hSCD1-inhibition.

Discovery of [(3-bromo-7-cyano-2-naphthyl)(difluoro)methyl]phosphonic acid, a potent and orally active small molecule PTP1B inhibitor.

A series of quinoline/naphthalene-difluoromethylphosphonates were prepared and were found to be potent PTP1B inhibitors. Most of these compounds bearing polar functionalities or large lipophilic residues did not show appreciable oral bioavailability in rodents while small and less polar analogs displayed moderate to good oral bioavailability. The title compound was found to have the best overall potency and pharmacokinetic profile and was found to be efficacious in animal models of diabetes and cancer.

Restoring methicillin-resistant Staphylococcus aureus susceptibility to ß-lactam antibiotics.

Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to ß-lactam antibiotics paired with ß-lactamase inhibitors. We have adapted a Staphylococcus aureus antisense knockdown strategy to genetically identify the cell division Z ring components-FtsA, FtsZ, and FtsW-as ß-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA). We demonstrate that the FtsZ-specific inhibitor PC190723 acts synergistically with ß-lactam antibiotics in vitro and in vivo and that this combination is efficacious in a murine model of MRSA infection. Fluorescence microscopy localization studies reveal that synergy between these agents is likely to be elicited by the concomitant delocalization of their cognate drug targets (FtsZ and PBP2) in MRSA treated with PC190723. A 2.0 Å crystal structure of S. aureus FtsZ in complex with PC190723 identifies the compound binding site, which corresponds to the predominant location of mutations conferring resistance to PC190723 (PC190723(R)). Although structural studies suggested that these drug resistance mutations may be difficult to combat through chemical modification of PC190723, combining PC190723 with the ß-lactam antibiotic imipenem markedly reduced the spontaneous frequency of PC190723(R) mutants. Multiple MRSA PC190723(R) FtsZ mutants also displayed attenuated virulence and restored susceptibility to ß-lactam antibiotics in vitro and in a mouse model of imipenem efficacy. Collectively, these data support a target-based approach to rationally develop synergistic combination agents that mitigate drug resistance and effectively treat MRSA infections.

High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1.

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 µM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.

Discovery of novel P2Y14 agonist and antagonist using conventional and nonconventional methods.

P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.

Development of a liver-targeted stearoyl-CoA desaturase (SCD) inhibitor (MK-8245) to establish a therapeutic window for the treatment of diabetes and dyslipidemia.

The potential use of SCD inhibitors for the chronic treatment of diabetes and dyslipidemia has been limited by preclinical adverse events associated with inhibition of SCD in skin and eye tissues. To establish a therapeutic window, we embarked on designing liver-targeted SCD inhibitors by utilizing molecular recognition by liver-specific organic anion transporting polypeptides (OATPs). In doing so, we set out to target the SCD inhibitor to the organ believed to be responsible for the therapeutic efficacy (liver) while minimizing its exposure in the tissues associated with mechanism-based SCD depletion of essential lubricating lipids (skin and eye). These efforts led to the discovery of MK-8245 (7), a potent, liver-targeted SCD inhibitor with preclinical antidiabetic and antidyslipidemic efficacy with a significantly improved therapeutic window.

Enzyme occupancy measurement of intracellular protein tyrosine phosphatase 1B using photoaffinity probes.

Protein tyrosine phosphatase 1B (PTP1B) is believed to be one of the enzymes involved in down-regulating the insulin receptor and is a drug target for the treatment of type II diabetes. To better understand the in vitro and in vivo behavior of PTP1B inhibitors, a cell-based assay to directly measure enzyme occupancy of PTP1B by inhibitors using photoaffinity labeling was developed. Two photoaffinity probes were synthesized containing the photolabile diazirine moiety. These photoprobes were specific for PTP1B and T-cell protein tyrosine phosphatase over CD45, with the most potent photoprobe having an IC(50) value of 0.2nM for PTP1B. Activation of the photoprobes with a 40-W UV lamp in the presence of purified AspTyrLysAspAspAspAspLys (Flag)-PTP1B formed a 1:1 irreversible adduct with the enzyme. The photolabeling was competed by known PTP1B inhibitors, vanadate, and the peptide inhibitor N-benzoyl-l-glutamyl-[4-phosphono(difluoromethyl)]-l-phenylalanyl-[4-phosphono(difluoromethyl)]l-phenylalanineamide (BzN-EJJ-amide). In HepG2 (human hepatoma cell line) cells, endogenous PTP1B was labeled by the UV-activated photoprobes in both lysed and intact cells. Enzyme occupancy measurements were conducted with a series of PTP1B inhibitors using the photoprobe affinity assay. Several compounds were shown to bind to endogenous PTP1B in the HepG2 intact cells.

Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding.

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

Protein tyrosine phosphatase: enzymatic assays.

Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.

Synthesis of a novel peptidic photoaffinity probe for the PTP-1B enzyme.

The synthesis of a novel radioactive peptidic photoaffinity probe for the PTP-1B enzyme as well as some SAR leading to the choice of this compound as a photoaffinity probe are presented.

The development of potent non-peptidic PTP-1B inhibitors.

The SAR from our peptide libraries was exploited to design a series of potent deoxybenzoin PTP-1B inhibitors. The introduction of an ortho bromo substituent next to the difluoromethylphosphonate warhead gave up to 20-fold increase in potency compared to the desbromo analogues. In addition, these compounds were orally bioavailable and active in the animal models of non-insulin dependent diabetes mellitus (NIDDM).

Structure based design of a series of potent and selective non peptidic PTP-1B inhibitors.

A series of benzotriazole phenyldifluoromethylphosphonic acids were found to be potent PTP-1B inhibitors. Molecular modeling on the X-ray crystal structure of the lead structure led to the design of potent PTP-1B inhibitors that show moderate selectivity against TC-PTP, a very closely related protein tyrosine phosphatase.

The structural basis for the selectivity of benzotriazole inhibitors of PTP1B.

Protein tyrosine phosphatase 1B (PTP1B) has been implicated in the regulation of the insulin signaling pathway and represents an attractive target for the design of inhibitors in the treatment of type 2 diabetes and obesity. Inspection of the structure of PTP1B indicates that potent PTP1B inhibitors may be obtained by targeting a secondary aryl phosphate-binding site as well as the catalytic site. We report here the crystal structures of PTP1B in complex with first and second generation aryldifluoromethyl-phosphonic acid inhibitors. While all compounds bind in a previously unexploited binding pocket near the primary binding site, the second generation compounds also reach into the secondary binding site, and exhibit moderate selectivity for PTP1B over the closely related T-cell phosphatase. The molecular basis for the selectivity has been confirmed by single point mutation at position 52, where the two phosphatases differ by a phenylalanine-to-tyrosine switch. These compounds present a novel platform for the development of potent and selective PTP1B inhibitors.