D-DOPA Is a Potent, Orally Bioavailable, Allosteric Inhibitor of Glutamate Carboxypeptidase II.

Glutamate carboxypeptidase-II (GCPII) is a zinc-dependent metalloenzyme implicated in numerous neurological disorders. The pharmacophoric requirements of active-site GCPII inhibitors makes them highly charged, manifesting poor pharmacokinetic (PK) properties. Herein, we describe the discovery and characterization of catechol-based inhibitors including L-DOPA, D-DOPA, and caffeic acid, with sub-micromolar potencies. Of these, D-DOPA emerged as the most promising compound, with good metabolic stability, and excellent PK properties. Orally administered D-DOPA yielded high plasma exposures (AUCplasma = 72.7 nmol·h/mL) and an absolute oral bioavailability of 47.7%. Unfortunately, D-DOPA brain exposures were low with AUCbrain = 2.42 nmol/g and AUCbrain/plasma ratio of 0.03. Given reports of isomeric inversion of D-DOPA to L-DOPA via D-amino acid oxidase (DAAO), we evaluated D-DOPA PK in combination with the DAAO inhibitor sodium benzoate and observed a >200% enhancement in both plasma and brain exposures (AUCplasma = 185 nmol·h/mL; AUCbrain = 5.48 nmol·h/g). Further, we demonstrated GCPII target engagement; orally administered D-DOPA with or without sodium benzoate caused significant inhibition of GCPII activity. Lastly, mode of inhibition studies revealed D-DOPA to be a noncompetitive, allosteric inhibitor of GCPII. To our knowledge, this is the first report of D-DOPA as a distinct scaffold for GCPII inhibition, laying the groundwork for future optimization to obtain clinically viable candidates.

Recent development in the discovery of PARP inhibitors as anticancer agents: a patent update (2016-2020).

INTRODUCTION: Discovery of small molecules that impede the activity of single-strand DNA repair enzyme, PARP1, has led to four marketed drugs for the treatment of advanced-stage cancers. Hence, there is a renewed enthusiasm in the PARP inhibitor discovery arena. To reduce nonspecific interactions or potential toxicities, and to understand the role of other minimally explored PARP enzymes, exciting new findings have emerged toward the development of selective inhibitors and targeted chemical biology probes. Importantly, the conventional PARP inhibitor design has evolved in a way that could potentially lead to multienzyme-targeting - a polypharmacological approach against aggressive cancers. AREAS COVERED: This review comprises recent progress made in the development of PARP inhibitors, primarily focused on human cancers. Discovery of novel PARP inhibitors with pan, selective, and multi-target inhibition using in vitro and in vivo cancer models is summarized and critically evaluated. Emphasis is given to patents published during 2016-2020, excluding TNKS 1/2 inhibitors. EXPERT OPINION: The outstanding success demonstrated by the FDA approved PARP inhibitors has fueled further clinical evaluations for expansion of their clinical utilities. The current clinical investigations include new candidates as well as marketed PARP-targeted drugs, both as single agents and in combination with other chemotherapeutics. Recent advances have also unveiled critical roles of other PARPs in oncogenic signal transduction, in addition to those of the well-documented PARP1/2 and TNKS1/2 enzymes. Further studies on lesser-known PARP members are urgently needed for functional annotations and for understanding their roles in cancer progression and other human diseases.

Structural and computational basis for potent inhibition of glutamate carboxypeptidase II by carbamate-based inhibitors.

A series of carbamate-based inhibitors of glutamate carboxypeptidase II (GCPII) were designed and synthesized using ZJ-43, N-[[[(1S)-1-carboxy-3-methylbutyl]amino]carbonyl]-l-glutamic acid, as a molecular template in order to better understand the impact of replacing one of the two nitrogen atoms in the urea-based GCPII inhibitor with an oxygen atom. Compound 7 containing a C-terminal 2-oxypentanedioic acid was more potent than compound 5 containing a C-terminal glutamic acid (2-aminopentanedioic acid) despite GCPII's preference for peptides containing an N-terminal glutamate as substrates. Subsequent crystallographic analysis revealed that ZJ-43 and its two carbamate analogs 5 and 7 with the same (S,S)-stereochemical configuration adopt a nearly identical binding mode while (R,S)-carbamate analog 8 containing a d-leucine forms a less extensive hydrogen bonding network. QM and QM/MM calculations have identified no specific interactions in the GCPII active site that would distinguish ZJ-43 from compounds 5 and 7 and attributed the higher potency of ZJ-43 and compound 7 to the free energy changes associated with the transfer of the ligand from bulk solvent to the protein active site as a result of the lower ligand strain energy and solvation/desolvation energy. Our findings underscore a broader range of factors that need to be taken into account in predicting ligand-protein binding affinity. These insights should be of particular importance in future efforts to design and develop GCPII inhibitors for optimal inhibitory potency.

D-Amino acid oxidase inhibitors based on the 5-hydroxy-1,2,4-triazin-6(1H)-one scaffold.

A series of 3-substituted 5-hydroxy-1,2,4-triazin-6(1H)-one derivatives were designed and synthesized as a new class of d-amino acid oxidase (DAAO) inhibitors. Some of the newly synthesized derivatives showed potent inhibitory activity against human DAAO with IC50 values in the nanomolar range. Among them, 6-hydroxy-3-phenethyl-1,2,4-triazin-5(2H)-one 6b and 3-((6-fluoronaphthalen-2-yl)methylthio)-6-hydroxy-1,2,4-triazin-5(2H)-one 6m were found to be metabolically stable in mouse liver microsomes. In addition, compound 6b was found to be orally available in mice and able to enhance plasma d-serine levels following its co-administration with d-serine compared to the oral administration of d-serine alone.

Peptidomimetics of Arg-Phe-NH2 as small molecule agonists of Mas-related gene C (MrgC) receptors.

A series of Arg-Phe-NH2 peptidomimetics containing an Arg mimetic were synthesized and tested as agonists of human MrgX1, rat MrgC, and mouse MrgC11 receptors. As predicted from the previously established species specificity, these peptidomimetics were found to be devoid of MrgX1 agonist activity. In contrast, these compounds acted as agonists of MrgC and/or MrgC11 with varying degrees of potency. These new peptidomimetics should complement the existing small molecule human MrgX1 agonists and enhance our ability to assess the therapeutic utility of targeting Mrg receptors in rodent models.

δ-Thiolactones as prodrugs of thiol-based glutamate carboxypeptidase II (GCPII) inhibitors.

δ-Thiolactones derived from thiol-based glutamate carboxypeptidase II (GCPII) inhibitors were evaluated as prodrugs. In rat liver microsomes, 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, 1) was gradually produced from 3-(2-oxotetrahydrothiopyran-3-yl)propionic acid (5), a thiolactone derived from 1. Compound 1 was detected in plasma at concentrations well above its IC50 for GCPII following oral administration of 5 in rats. Consistent with the oral plasma pharmacokinetics, thiolactone 5 exhibited efficacy in a rat model of neuropathic pain following oral administration.

Kinetic characterization of ebselen, chelerythrine and apomorphine as glutaminase inhibitors.

Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC(1280))) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.

Synthesis of kojic acid derivatives as secondary binding site probes of D-amino acid oxidase.

A series of kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) derivatives were synthesized and tested for their ability to inhibit D-amino acid oxidase (DAAO). Various substituents were incorporated into kojic acid at its 2-hydroxymethyl group. These analogs serve as useful molecular probes to explore the secondary binding site, which can be exploited in designing more potent DAAO inhibitors.

Synthesis and SAR of 1-hydroxy-1H-benzo[d]imidazol-2(3H)-ones as Inhibitors of D-Amino Acid Oxidase.

A series of 1-hydroxy-1H-benzo[d]imidazol-2(3H)-ones were synthesized and evaluated for their ability to inhibit human and porcine forms of D-amino acid oxidase (DAAO). Inhibitory potency is largely dependent on the size and position of substituents on the benzene ring with IC(50) values of the compounds ranging from 70 nM to greater than 100 µM. Structure-activity relationships of this new class of DAAO inhibitors will be presented in detail along with comparisons to previously published SAR data from other classes of DAAO inhibitors. Some of these compounds were given to mice orally together with D-serine to assess their effects on plasma D-serine pharmacokinetics.

Design, synthesis, and pharmacological evaluation of bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) analogs as glutaminase inhibitors.

Bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) is a potent and selective allosteric inhibitor of kidney-type glutaminase (GLS) that has served as a molecular probe to determine the therapeutic potential of GLS inhibition. In an attempt to identify more potent GLS inhibitors with improved drug-like molecular properties, a series of BPTES analogs were synthesized and evaluated. Our structure-activity relationship (SAR) studies revealed that some truncated analogs retained the potency of BPTES, presenting an opportunity to improve its aqueous solubility. One of the analogs, N-(5-{2-[2-(5-amino-[1,3,4]thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]thiadiazol-2-yl)-2-phenyl-acetamide 6, exhibited similar potency and better solubility relative to BPTES and attenuated the growth of P493 human lymphoma B cells in vitro as well as in a mouse xenograft model.

Design, synthesis, and pharmacological evaluation of glutamate carboxypeptidase II (GCPII) inhibitors based on thioalkylbenzoic acid scaffolds.

A series of thiol-based glutamate carboxypeptidase II (GCPII) inhibitors have been synthesized with either a 3-(mercaptomethyl)benzoic acid or 2-(2-mercaptoethyl)benzoic acid scaffold. Potent inhibitors were identified from each of the two scaffolds with IC(50) values in the single-digit nanomolar range, including 2-(3-carboxybenzyloxy)-5-(mercaptomethyl)benzoic acid 27c and 3-(2-mercaptoethyl)biphenyl-2,3'-dicarboxylic acid 35c. Compound 35c was found to be metabolically stable and selective over a number of targets related to glutamate-mediated neurotransmission. Furthermore, compound 35c was found to be orally available in rats and exhibited efficacy in an animal model of neuropathic pain following oral administration.

The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type.

The altered metabolism of tumors has been considered a target for anticancer therapy. However, the relationship between distinct tumor-initiating lesions and anomalies of tumor metabolism in vivo has not been addressed. We report that MYC-induced mouse liver tumors significantly increase both glucose and glutamine catabolism, whereas MET-induced liver tumors use glucose to produce glutamine. Increased glutamine catabolism in MYC-induced liver tumors is associated with decreased levels of glutamine synthetase (Glul) and the switch from Gls2 to Gls1 glutaminase. In contrast to liver tumors, MYC-induced lung tumors display increased expression of both Glul and Gls1 and accumulate glutamine. We also show that inhibition of Gls1 kills cells that overexpress MYC and catabolize glutamine. Our results suggest that the metabolic profiles of tumors are likely to depend on both the genotype and tissue of origin and have implications regarding the design of therapies targeting tumor metabolism.

Inhibition of xc⁻ transporter-mediated cystine uptake by sulfasalazine analogs.

A series of sulfasalazine analogs were synthesized and tested for their ability to block cystine-glutamate antiporter system xc⁻ using L-[(14)C]cystine as a substrate. Replacement of sulfasalazine's diazo group with an alkyne group led to an equally potent inhibitor, 2-hydroxy-5-((4-(N-pyridin-2-ylsulfamoyl)phenyl)ethynyl)benzoic acid 6. Our SAR studies also revealed that the carboxylate group of sulfasalazine is essential for its inhibitory activity while the phenolic hydroxyl group is dispensable. Truncated analogs lacking an N-pyridin-2-ylsulfamoyl moiety were less potent than sulfasalazine, but may serve as more tractable templates because of their low molecular weight by applying a variety of fragment growing approaches. Given that sulfasalazine is rapidly metabolized through cleavage of the diazo bond, these analogs may possess a more desirable pharmacological profile as system xc- blockers, in particular, for in vivo studies.

Recent advances in the discovery of D-amino acid oxidase inhibitors and their therapeutic utility in schizophrenia.

D-amino acid oxidase (DAAO) catalyzes the oxidative metabolism of D-amino acids including D-serine, a full agonist at the allosteric glycine binding site of the NMDA receptor. D-serine was reported to improve negative and cognitive symptoms of schizophrenia, symptoms poorly addressed by the standard D2 antagonist therapies. Therefore, inhibition of DAAO has gained substantial interest as an effective way to increase D-serine levels in the brain. During the last several years, a growing number of structurally diverse DAAO inhibitors have been identified with significantly higher inhibitory potency compared to the conventional DAAO inhibitors. Some of these new generation of DAAO inhibitors are being evaluated for their ability to enhance D-serine levels in rodents and efficacy in animal models of schizophrenia. This article highlights the progress that has been made toward the discovery of DAAO inhibitors and recent efforts to exploit their therapeutic utility in schizophrenia.

Inhibition of glutaminase preferentially slows growth of glioma cells with mutant IDH1.

Mutation at the R132 residue of isocitrate dehydrogenase 1 (IDH1), frequently found in gliomas and acute myelogenous leukemia, creates a neoenzyme that produces 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). We sought to therapeutically exploit this neoreaction in mutant IDH1 cells that require α-KG derived from glutamine. Glutamine is converted to glutamate by glutaminase and further metabolized to α-KG. Therefore, we inhibited glutaminase with siRNA or the small molecule inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and found slowed growth of glioblastoma cells expressing mutant IDH1 compared with those expressing wild-type IDH1. Growth suppression of mutant IDH1 cells by BPTES was rescued by adding exogenous α-KG. BPTES inhibited glutaminase activity, lowered glutamate and α-KG levels, and increased glycolytic intermediates while leaving total 2-HG levels unaffected. The ability to selectively slow growth in cells with IDH1 mutations by inhibiting glutaminase suggests a unique reprogramming of intermediary metabolism and a potential therapeutic strategy.

The discovery and structure-activity relationships of indole-based inhibitors of glutamate carboxypeptidase II.

A series of N-substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acids were synthesized as inhibitors of glutamate carboxypeptidase II (GCPII). Those containing carboxybenzyl or carboxyphenyl groups at the N-position exhibited potent inhibitory activity against GCPII. These indole-based compounds represent the first example of achiral GCPII inhibitors and demonstrate greater tolerance of the GCPII active site for ligands with significant structural difference from the endogenous substrate, N-acetyl-aspartylglutamate.

Novel mechanism of inhibition of rat kidney-type glutaminase by bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES).

The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide]. BPTES is a potent inhibitor of KGA, but not of the liver GA isoform, glutamate dehydrogenase or gamma-glutamyl transpeptidase. Kinetic studies indicate that, with respect to glutamine, BPTES has a K(i) of approx. 3 microM. Moreover, these studies suggest that BPTES inhibits the allosteric activation caused by phosphate binding and promotes the formation of an inactive complex. Gel-filtration chromatography and sedimentation-velocity analysis were used to examine the effect of BPTES on the phosphate-dependent oligomerization of KGA. This established that BPTES prevents the formation of large phosphate-induced oligomers and instead promotes the formation of a single oligomeric species with distinct physical properties. Sedimentation-equilibrium studies determined that the oligomer produced by BPTES is a stable tetramer. Taken together, the present work indicates that BPTES is a unique and potent inhibitor of rat KGA and elucidates a novel mechanism of inactivation.