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.

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 IC values of 1.

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.

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

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-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.

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

Despite the need for new antibiotics to treat drug-resistant bacteria, current clinical combinations are largely restricted to β-lactam antibiotics paired with β-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 β-lactam susceptibility determinants of methicillin-resistant S. aureus (MRSA).

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically inactive (C215S) mutant.

Protein tyrosine phosphatases are a class of enzymes that function to modulate tyrosine phosphorylation of cellular proteins and play an essential role in regulating cell function. PTP1B has been implicated in the negative regulation of the insulin signaling pathway by dephosphorylating the activated insulin receptor. Inhibiting this phosphatase and preventing the insulin-receptor downregulation has been suggested as a target for the treatment of Type II diabetes.

The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase.

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48--50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP.

A two-component affinity chromatography purification of Helix pomatia arylsulfatase by tyrosine vanadate.

The inhibition of Helix pomatia arylsulfatase by the synergistic combination of N-acetyl-l-tyrosine ethyl ester and vanadate has been extended to affinity chromatography for purification. In the presence of vanadate, l-tyrosine ethyl ester (TEE), immobilized on CH-Sepharose 4B retained arylsulfatase from the digestive juice or lyophilized powder of H. pomatia.

Calcium is not required for 5-lipoxygenase activity at high phosphatidyl choline vesicle concentrations.

5-Lipoxygenase (5-LO) catalyzes the formation of 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) and leukotriene A4 (LTA4) from arachidonic acid. Following a rise in intracellular calcium, 5-LO translocates to a membrane where it reacts with arachidonic acid via an 18 kD protein (FLAP). In vitro studies using a vesicle system of phosphatidylcholine (PC) and purified 5-LO were conducted under varying concentrations of PC and calcium.

How does alendronate inhibit protein-tyrosine phosphatases?

Alendronate (4-amino-1-hydroxybutylidene 1,1-bisphosphonate) is a drug used in the treatment of osteoporosis and other bone diseases. The inhibition of protein-tyrosine phosphatases (PTPs) by alendronate suggests that PTPs may be molecular targets. As a clear understanding of the inhibition mechanism is lacking, our aim was to analyze the mechanism to provide further insight into its therapeutic effect.

Characterization of autocrine inducible prostaglandin H synthase-2 (PGHS-2) in human osteosarcoma cells.

The human osteosarcoma 143.98.2 cell line was found to express high levels of prostaglandin synthase-2 (PGHS-2) without detectable levels of prostaglandin synthase-1 (PGHS-1) as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis.