Allosteric kidney-type glutaminase (GLS) inhibitors with a mercaptoethyl linker.

A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a mercaptoethyl (SCH2CH2) linker were synthesized in an effort to further expand the structural diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl linker between the two thiadiazole rings was found to potently inhibit GLS with an IC50 value of 50 nM. Interestingly, the corresponding derivative with an n-propyl (CH2CH2CH2) linker showed substantially lower inhibitory potency (IC50 = 2.3 µM) while the derivative with a dimethylsulfide (CH2SCH2) linker showed no inhibitory activity at concentrations up to 100 µM, underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and tested as GLS inhibitors to further explore SAR within this scaffold including derivatives possessing a pyridazine as a replacement for one of the two thiadiazole moiety.

Recent Progress in the Discovery of Allosteric Inhibitors of Kidney-Type Glutaminase.

Kidney-type glutaminase (GLS), the first enzyme in the glutaminolysis pathway, catalyzes the hydrolysis of glutamine to glutamate. GLS was found to be upregulated in many glutamine-dependent cancer cells. Therefore, selective inhibition of GLS has gained substantial interest as a therapeutic approach targeting cancer metabolism. Bis-2-[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide (BPTES), despite its poor physicochemical properties, has served as a key molecular template in subsequent efforts to identify more potent and drug-like allosteric GLS inhibitors. This review article provides an overview of the progress made to date in the development of GLS inhibitors and highlights the remarkable transformation of the unfavorable lead into "druglike" compounds guided by systematic SAR studies.

DPTIP, a newly identified potent brain penetrant neutral sphingomyelinase 2 inhibitor, regulates astrocyte-peripheral immune communication following brain inflammation.

Brain injury and inflammation induces a local release of extracellular vesicles (EVs) from astrocytes carrying proteins, RNAs, and microRNAs into the circulation. When these vesicles reach the liver, they stimulate the secretion of cytokines that mobilize peripheral immune cell infiltration into the brain, which can cause secondary tissue damage and impair recovery. Recent studies suggest that suppression of EV biosynthesis through neutral sphingomyelinase 2 (nSMase2) inhibition may represent a new therapeutic strategy. Unfortunately, currently available nSMase2 inhibitors exhibit low potency (IC50 ≥ 1 µM), poor solubility and/or limited brain penetration. Through a high throughput screening campaign of >365,000 compounds against human nSMase2 we identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), a potent (IC50 30 nM), selective, metabolically stable, and brain penetrable (AUCbrain/AUCplasma = 0.26) nSMase2 inhibitor. DPTIP dose-dependently inhibited EV release in primary astrocyte cultures. In a mouse model of brain injury conducted in GFAP-GFP mice, DPTIP potently (10 mg/kg IP) inhibited IL-1ß-induced astrocyte-derived EV release (51 ± 13%; p < 0.001). This inhibition led to a reduction of cytokine upregulation in liver and attenuation of the infiltration of immune cells into the brain (80 ± 23%; p < 0.01). A structurally similar but inactive analog had no effect in vitro or in vivo.

N-Substituted Prodrugs of Mebendazole Provide Improved Aqueous Solubility and Oral Bioavailability in Mice and Dogs.

Mebendazole (MBZ) was developed as a broad-spectrum anthelmintic but has recently shown efficacy as an anticancer agent. The use of MBZ for cancer, however, is challenging due to its poor solubility leading to poor bioavailability. Herein, we developed a prodrug approach with various N-linked promoieties including acyloxymethyl, aminoacyloxymethyl, and substituted phosphonooxymethyl in attempt to improve these characteristics. Compound 12, containing an (((((isopropoxycarbonyl)oxy)methoxy)phosphoryl)oxy)methyl promoiety, showed a >10 000-fold improvement in aqueous solubility. When evaluated in mice, 12 displayed a 2.2-fold higher plasma AUC0- t and a 1.7-fold improvement in brain AUC0- t with a calculated oral bioavailability of 52%, as compared to 24% for MBZ-polymorph C (MBZ-C), the most bioavailable polymorph. In dogs, 12 showed a 3.8-fold higher plasma AUC0- t with oral bioavailability of 41% compared to 11% for MBZ-C. In summary, we have identified a prodrug of MBZ with better physicochemical properties and enhanced bioavailability in both mice and dog.

Enhanced Brain Delivery of 2-(Phosphonomethyl)pentanedioic Acid Following Intranasal Administration of Its γ-Substituted Ester Prodrugs.

2-(Phosphonomethyl)pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase-II (GCPII) with efficacy in multiple neurological and psychiatric disease models, but its clinical utility is hampered by low brain penetration due to the inclusion of multiple acidic functionalities. We recently reported an improvement in the brain-to-plasma ratio of 2-PMPA after intranasal (IN) dosing in both rodents and primates. Herein, we describe the synthesis of several 2-PMPA prodrugs with further improved brain delivery of 2-PMPA after IN administration by masking of the γ-carboxylate. When compared to IN 2-PMPA in rats at 1 h post dose, γ-(4-acetoxybenzyl)-2-PMPA (compound 1) resulted in significantly higher 2-PMPA delivery to both plasma (4.1-fold) and brain (11-fold). Subsequent time-dependent evaluation of 1 also showed high brain as well as plasma 2-PMPA exposures with brain-to-plasma ratios of 2.2, 0.48, and 0.26 for olfactory bulb, cortex, and cerebellum, respectively, as well as an improved sciatic nerve to plasma ratio of 0.84. In contrast, IV administration of compound 1 resulted in similar plasma exposure of 2-PMPA versus the IN route (AUCIV: 76 ± 9 h·nmol/mL versus AUCIN: 99 ± 24 h·nmol/mL); but significantly lower nerve and brain tissue exposures with tissue-to-plasma ratios of 0.21, 0.03, 0.04, and 0.04 in nerve, olfactory bulb, cortex, and cerebellum, respectively. In primates, IN administration of 1 more than doubled 2-PMPA concentrations in the cerebrospinal fluid relative to previously reported levels following IN 2-PMPA. The results of these experiments provide a promising strategy for testing GCPII inhibition in neurological and psychiatric disorders.

Discovery of a para-Acetoxy-benzyl Ester Prodrug of a Hydroxamate-Based Glutamate Carboxypeptidase II Inhibitor as Oral Therapy for Neuropathic Pain.

4-Carboxy-α-[3-(hydroxyamino)-3-oxopropyl]-benzenepropanoic acid 1 is a potent hydroxamate-based inhibitor of glutamate carboxypeptidase II. In an attempt to improve its poor oral pharmacokinetics, we synthesized a series of prodrugs by masking its hydrophilic hydroxamate group. Prodrugs were evaluated for oral availability in mice and showed varying degree of plasma exposure to 1. Of these, para-acetoxybenzyl-based, 4-(5-(((4-acetoxybenzyl)oxy)amino)-2-carboxy-5-oxopentyl)benzoic acid, 12, provided 5-fold higher plasma levels of 1 compared to oral administration of 1 itself. Subsequently, para-acetoxybenzyl-based prodrugs with additional ester promoiety(ies) on carboxylate(s) were examined for their ability to deliver 1 to plasma. Isopropyloxycarbonyloxymethyl (POC) ester 30 was the only prodrug that achieved substantial plasma levels of 1. In vitro metabolite identification studies confirmed stability of the ethyl ester of benzoate while the POC group was rapidly hydrolyzed. At oral daily dose-equivalent of 3 mg/kg, 12 exhibited analgesic efficacy comparable to dose of 10 mg/kg of 1 in the rat chronic constrictive injury model of neuropathic pain.

N-(Pivaloyloxy)alkoxy-carbonyl Prodrugs of the Glutamine Antagonist 6-Diazo-5-oxo-l-norleucine (DON) as a Potential Treatment for HIV Associated Neurocognitive Disorders.

Aberrant excitatory neurotransmission associated with overproduction of glutamate has been implicated in the development of HIV-associated neurocognitive disorders (HAND). The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 14) attenuates glutamate synthesis in HIV-infected microglia/macrophages, offering therapeutic potential for HAND. We show that 14 prevents manifestation of spatial memory deficits in chimeric EcoHIV-infected mice, a model of HAND. 14 is not clinically available, however, because its development was hampered by peripheral toxicities. We describe the synthesis of several substituted N-(pivaloyloxy)alkoxy-carbonyl prodrugs of 14 designed to circulate inert in plasma and be taken up and biotransformed to 14 in the brain. The lead prodrug, isopropyl 6-diazo-5-oxo-2-(((phenyl(pivaloyloxy)methoxy)carbonyl)amino)hexanoate (13d), was stable in swine and human plasma but liberated 14 in swine brain homogenate. When dosed systemically in swine, 13d provided a 15-fold enhanced CSF-to-plasma ratio and a 9-fold enhanced brain-to-plasma ratio relative to 14, opening a possible clinical path for the treatment of HAND.

Local enema treatment to inhibit FOLH1/GCPII as a novel therapy for inflammatory bowel disease.

Here we evaluate the potential for local administration of a small molecule FOLH1/GCPII inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) as a novel treatment for inflammatory bowel disease (IBD). We found that FOLH1/GCPII enzyme activity was increased in the colorectal tissues of mice with TNBS-induced colitis, and confirmed that 2-PMPA inhibited FOLH1/GCPII enzyme activity ex vivo. In order to maximize local enema delivery of 2-PMPA, we studied the effect of vehicle tonicity on the absorption of 2-PMPA in the colon. Local administration of 2-PMPA in a hypotonic enema vehicle resulted in increased colorectal tissue absorption at 30min compared to 2-PMPA administered in an isotonic enema vehicle. Furthermore, local delivery of 2-PMPA in hypotonic enema vehicle resulted in prolonged drug concentrations for at least 24h with minimal systemic exposure. Finally, daily treatment with the hypotonic 2-PMPA enema ameliorated macroscopic and microscopic symptoms of IBD in the TNBS-induced colitis mouse model, indicating the potential of FOLH1/GCPII inhibitors for the local treatment of IBD.

Combination therapy with BPTES nanoparticles and metformin targets the metabolic heterogeneity of pancreatic cancer.

Targeting glutamine metabolism via pharmacological inhibition of glutaminase has been translated into clinical trials as a novel cancer therapy, but available drugs lack optimal safety and efficacy. In this study, we used a proprietary emulsification process to encapsulate bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a selective but relatively insoluble glutaminase inhibitor, in nanoparticles. BPTES nanoparticles demonstrated improved pharmacokinetics and efficacy compared with unencapsulated BPTES. In addition, BPTES nanoparticles had no effect on the plasma levels of liver enzymes in contrast to CB-839, a glutaminase inhibitor that is currently in clinical trials. In a mouse model using orthotopic transplantation of patient-derived pancreatic tumor tissue, BPTES nanoparticle monotherapy led to modest antitumor effects. Using the HypoxCR reporter in vivo, we found that glutaminase inhibition reduced tumor growth by specifically targeting proliferating cancer cells but did not affect hypoxic, noncycling cells. Metabolomics analyses revealed that surviving tumor cells following glutaminase inhibition were reliant on glycolysis and glycogen synthesis. Based on these findings, metformin was selected for combination therapy with BPTES nanoparticles, which resulted in significantly greater pancreatic tumor reduction than either treatment alone. Thus, targeting of multiple metabolic pathways, including effective inhibition of glutaminase by nanoparticle drug delivery, holds promise as a novel therapy for pancreatic cancer.

Structural basis for exploring the allosteric inhibition of human kidney type glutaminase.

Cancer cells employ glutaminolysis to provide a source of intermediates for their upregulated biosynthetic needs. Glutaminase, which catalyzes the conversion of glutamine to glutamate, is gaining increasing attention as a potential drug target. Small-molecule inhibitors such as BPTES and CB-839, which target the allosteric site of glutaminase with high specificity, demonstrate immense promise as anti-tumor drugs. Here, we report the study of a new BPTES analog, N,N'-(5,5'-(trans-cyclohexane-1,3-diyl)bis(1,3,4-tiadiazole-5,2-diyl))bis(2-phenylacetamide) (trans-CBTBP), and compared its inhibitory effect against that of CB-839 and BPTES. We show that CB-839 has a 30- and 50-fold lower IC50 than trans-CBTBP and BPTES, respectively. To explore the structural basis for the differences in their inhibitory efficacy, we solved the complex structures of cKGA with 1S, 3S-CBTBP and CB-839. We found that CB-839 produces a greater degree of interaction with cKGA than 1S, 3S-CBTBP or BPTES. The results of this study will facilitate the rational design of new KGA inhibitors to better treat glutamine-addicted cancers.

Allosteric Glutaminase Inhibitors Based on a 1,4-Di(5-amino-1,3,4-thiadiazol-2-yl)butane Scaffold.

A series of allosteric kidney-type glutaminase (GLS) inhibitors were designed and synthesized using 1,4-di(5-amino-1,3,4-thiadiazol-2-yl)butane as a core scaffold. A variety of modified phenylacetyl groups were incorporated into the 5-amino group of the two thiadiazole rings in an attempt to facilitate additional binding interactions with the allosteric binding site of GLS. Among the newly synthesized compounds, 4-hydroxy-N-[5-[4-[5-[(2-phenylacetyl)amino]-1,3,4-thiadiazol-2-yl]butyl]-1,3,4-thiadiazol-2-yl]-benzeneacetamide, 2m, potently inhibited GLS with an IC50 value of 70 nM, although it did not exhibit time-dependency as seen with CB-839. Antiproliferative effects of 2m on human breast cancer lines will be also presented in comparison with those observed with CB-839.

Design and synthesis of a series of truncated neplanocin fleximers.

In an effort to study the effects of flexibility on enzyme recognition and activity, we have developed several different series of flexible nucleoside analogues in which the purine base is split into its respective imidazole and pyrimidine components. The focus of this particular study was to synthesize the truncated neplanocin A fleximers to investigate their potential anti-protozoan activities by inhibition of S-adenosylhomocysteine hydrolase (SAHase). The three fleximers tested displayed poor anti-trypanocidal activities, with EC50 values around 200 µM. Further studies of the corresponding ribose fleximers, most closely related to the natural nucleoside substrates, revealed low affinity for the known T. brucei nucleoside transporters P1 and P2, which may be the reason for the lack of trypanocidal activity observed.

Structure-Metabolism Relationships in the Glucuronidation of d-Amino Acid Oxidase Inhibitors.

Representative d-amino acid oxidase (DAAO) inhibitors were subjected to in vitro liver microsomal stability tests in the absence or presence of uridine diphosphate glucuronic acid (UDPGA). While carboxylate-based DAAO inhibitors displayed little glucuronidation, most DAAO inhibitors containing α-hydroxycarbonyl moiety exhibited nearly complete glucuronidation within 30 min. The one exception was 6-[2-(3,5-difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one 10, which exhibited some degree of resistance to glucuronidation by liver microsomes from mice, rats, and humans.

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.

Antiproliferative activities of halogenated thieno[3,2-d]pyrimidines.

The in vitro evaluation of thieno[3,2-d]pyrimidines identified halogenated compounds 1 and 2 with antiproliferative activity against three different cancer cell lines. A structure activity relationship study indicated the necessity of the chlorine at the C4-position for biological activity. The two most active compounds 1 and 2 were found to induce apoptosis in the leukemia L1210 cell line. Additionally, the compounds were screened against a variety of other microbial targets and as a result, selective activity against several fungi was also observed. The synthesis and preliminary biological results are reported herein.

"Reverse" carbocyclic fleximers: synthesis of a new class of adenosine deaminase inhibitors.

A series of flexible carbocyclic pyrimidine nucleosides has been designed and synthesized. In contrast to previously reported "fleximers" from our laboratory, these analogues have the connectivity of the heterocyclic base system "reversed", where the pyrimidine ring is attached to the sugar moiety, rather than the five membered imidazole ring. As was previously seen with the ribose fleximers, their inherent flexibility should allow them to adjust to enzyme binding site mutations, as well as increase the affinity for atypical enzymes. Preliminary biological screening has revealed surprising inhibition of adenosine deaminase, despite their lack of resemblance to adenosine.

Carbocyclic 5'-nor "reverse" fleximers. Design, synthesis, and preliminary biological activity.

A series of 5'-nor carbocyclic "reverse" flexible nucleosides or "fleximers" have been designed wherein the nucleobase scaffold resembles a "split" purine as well as a substituted pyrimidine. This modification was employed to explore recognition by both purine and pyrimidine metabolizing enzymes. The synthesis of the carbocyclic fleximers and the results of their preliminary biological screening are described herein.

Hetero-expanded purine nucleosides. Design, synthesis and preliminary biological activity.

Several thieno-expanded purine nucleoside analogues were synthesized for use as tools in ongoing investigations into nucleic acid structure and function in our laboratories. The inclusion of the thiophene ring system in the nucleoside endows the purine scaffold with advantages not previously available in other reported expanded purines. The synthesis and preliminary biological studies are reported herein.