In Vivo Efficacy of Relebactam (MK-7655) in Combination with Imipenem-Cilastatin in Murine Infection Models.

The World Health Organization has identified antimicrobial resistance as a global public health threat since the prevalence and spread of antibiotic resistance among bacterial pathogens worldwide are staggering. Carbapenems, such as imipenem and meropenem, have been used to treat multidrug-resistant bacteria; however, since the development of resistance to carbapenems, ß-lactam antibiotics in combination with ß-lactamase inhibitors (BLI) has been one of the most successful strategies to enhance the activity of ß-lactam antibiotics. Relebactam (REL) is a new BLI which has been found to inhibit class A and class C ß-lactamases in vitro REL has been reported to restore imipenem's activity against both imipenem-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae Reported here are the in vivo efficacy studies of the imipenem-cilastatin (IMI)-REL combination in mouse models of disseminated and pulmonary infection caused by imipenem-resistant clinical isolates of P. aeruginosa and K. pneumoniae The combination was also evaluated in a P. aeruginosa delayed pulmonary model of infection. IMI-REL was found to be effective in the disseminated model of infection with log reduction in P. aeruginosa CFU of 3.73, 3.13, and 1.72 at REL doses of 40, 20, and 10 mg/kg, respectively. For K. pneumoniae, log reductions in CFU of 2.36, 3.06, and 2.29 were reported at REL doses of 80, 40, and 20 mg/kg, respectively. The combination was less effective in the delayed pulmonary model than in the immediate pulmonary model; however, overall REL was found to be effective against these imipenem-resistant strains.

Design, Synthesis, and Evaluation of Novel and Selective G-protein Coupled Receptor 120 (GPR120) Spirocyclic Agonists.

Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effective insulin sensitizers, absent of weight gain, has been a long-standing goal of diabetes research. G-protein coupled receptor 120 (GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic agonists have been associated with insulin sensitizing, anti-inflammatory, and fat metabolism effects. Herein we describe the design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance (HOMA-IR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp in diet-induced obese (DIO) mice.

Discovery and Optimization of a Novel Triazole Series of GPR142 Agonists for the Treatment of Type 2 Diabetes.

GPR142 has been identified as a potential glucose-stimulated insulin secretion (GSIS) target for the treatment of type 2 diabetes mellitus (T2DM). A class of triazole GPR142 agonists was discovered through a high throughput screen. The lead compound 4 suffered from poor metabolic stability and poor solubility. Lead optimization strategies to improve potency, efficacy, metabolic stability, and solubility are described. This optimization led to compound 20e, which showed significant reduction of glucose excursion in wild-type but not in GPR142 deficient mice in an oral glucose tolerance test (oGTT) study. These studies provide strong evidence that reduction of glucose excursion through treatment with 20e is GPR142-mediated, and GPR142 agonists could be used as a potential treatment for type 2 diabetes.

Discovery of benzofuran propanoic acid GPR120 agonists: From uHTS hit to mechanism-based pharmacodynamic effects.

The transformation of an aryloxybutanoic acid ultra high-throughput screening (uHTS) hit into a potent and selective series of G-protein coupled receptor 120 (GPR120) agonists is reported. uHTS hit 1 demonstrated an excellent rodent pharmacokinetic profile and selectivity over the related fatty acid receptor GPR40, but only modest GPR120 potency. Optimization of the "left-hand" aryl group led to compound 6, which demonstrated a GPR120 mechanism-based pharmacodynamic effect in a mouse oral glucose tolerance test (oGTT). Further optimization gave rise to the benzofuran propanoic acid series (exemplified by compound 37), which demonstrated acute mechanism-based pharmacodynamic effects. The combination of in vivo efficacy and attractive rodent pharmacodynamic profiles suggests compounds generated from this series may afford attractive candidates for the treatment of Type 2 diabetes.

Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors.

Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

Discovery of Novel Tricyclic Heterocycles as Potent and Selective DPP-4 Inhibitors for the Treatment of Type 2 Diabetes.

In our efforts to develop second generation DPP-4 inhibitors, we endeavored to identify distinct structures with long-acting (once weekly) potential. Taking advantage of X-ray cocrystal structures of sitagliptin and other DPP-4 inhibitors, such as alogliptin and linagliptin bound to DPP-4, and aided by molecular modeling, we designed several series of heterocyclic compounds as initial targets. During their synthesis, an unexpected chemical transformation provided a novel tricyclic scaffold that was beyond our original design. Capitalizing on this serendipitous discovery, we have elaborated this scaffold into a very potent and selective DPP-4 inhibitor lead series, as highlighted by compound 17c.

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.

MK-4815, a potential new oral agent for treatment of malaria.

Malaria continues to have a significant impact on the health of the developing world. Efforts to combat this disease now focus on combination therapy in order to stem the emergence of resistant parasites. Continued efforts are needed to discover and develop new agents for use in combination antimalarial regimens. MK-4815 is a small molecule with antimalarial activity that was identified from a large pharmaceutical compound collection using a semiautomated version of a well-established in vitro assay for the erythrocytic stages of Plasmodium falciparum. In vitro studies indicate that the compound selectively accumulates in infected red blood cells and is most effective against the metabolically active late trophozoite/early schizont stages. A variety of drug-resistant field isolates of P. falciparum were found to be as sensitive to MK-4815 as the wild-type lines. MK-4815 is orally active in a P. berghei mouse model of acute malaria. In this model, where untreated animals succumb to infection 10 to 12 days postinfection, MK-4815 was completely curative when given as a single dose of 50 mg/kg, 2 doses of 25 mg/kg, or 4.5 doses of 12.5 mg/kg. In pharmacokinetic studies with mice and rhesus monkeys, MK-4815 demonstrated oral bioavailability and low clearance. In addition, MK-4815 is inexpensive to synthesize, an important characteristic for providing affordable antimalaria therapy to the developing world. The attractive biological and pharmaceutical profile of MK-4815 demonstrates its potential for use in combination with other agents in the fight against malaria.

Efficacy of caspofungin in a juvenile mouse model of central nervous system candidiasis.

Neonatal candidiasis is an increasingly common occurrence causing significant morbidity and mortality and a higher risk of dissemination to the central nervous system (CNS) than that seen with older patients. The current understanding of optimal antifungal therapy in this setting is limited. We have developed a model of disseminated candidiasis with CNS involvement in juvenile mice to assess the efficacy of the echinocandin caspofungin relative to amphotericin B (AmB). Juvenile mice were inoculated intravenously with 5.64 × 10(4) CFU of Candida albicans MY1055. Treatment with caspofungin at 1, 2, 4, and 8 mg/kg of body weight/day, AmB at 1 mg/kg/day, or a vehicle control (VC) was initiated 30 h after infection and continued for 7 days. Pharmacokinetic parameters for caspofungin were also determined. Culture and histology showed evidence of disseminated candidiasis with multifocal encephalitis at the start of antifungal therapy. Survival was 100% in all treated groups, while mortality was 100% in the VC by day 11 after infection. By day 5, all mice in the caspofungin treatment (four doses) groups showed reductions in kidney and brain burden relative to the VC, while AmB treatment reduced kidney burden but gave no reduction of brain fungal burden. Systemic levels of caspofungin were similar in infected and uninfected mice, while brain levels were higher in infected animals. In this juvenile mouse model, caspofungin demonstrated dose-dependent activity, equivalent to or better than that of AmB at 1 mg/kg, against disseminated candidiasis with CNS involvement.

Isolation and structure elucidation of parnafungins, antifungal natural products that inhibit mRNA polyadenylation.

The Candida albicans Fitness Test, a whole-cell screening platform, was used to profile crude fermentation extracts for novel antifungal natural products with interesting mechanisms of action. An extract with intrinsic antifungal activity from the fungus Fusarium larvarum displayed a Fitness Test profile that strongly implicated mRNA processing as the molecular target responsible for inhibition of fungal growth. Isolation of the active components from this sample identified a novel class of isoxazolidinone-containing natural products, which we have named parnafungins. These natural products were isolated as an interconverting mixture of four structural- and stereoisomers. The isomerization of the parnafungins was due to a retro-Michael ring-opening and subsequent reformation of a xanthone ring system. This interconversion was blocked by methylation of an enol moiety. Structure elucidation of purified parnafungin derivatives was accomplished by X-ray crystallography and NMR analysis. The biochemical target of these natural products has been identified as the fungal polyadenosine polymerase. Parnafungins demonstrated broad spectrum antifungal activity with no observed activity against gram-positive or gram-negative bacteria. The intact isoxazolidinone ring was required for antifungal activity. In addition, the natural products were efficacious in a mouse model of disseminated candidiasis.

Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): antiparasitic activity of a PKG inhibitor.

Cyclic GMP-dependent protein kinase (PKG) has been biochemically and genetically validated in Toxoplasma gondii as a primary target responsible for the antiparasitic activity of the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (Compound 1) [Biftu T, Feng D, Ponpipom M, et al. Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents. Bioorg Med Chem Lett 2005;15:3296-301; Gurnett AM, Liberator PA, Dulski PM, et al. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J Biol Chem 2002;277:15913-22; Donald RGK, Allocco J, Singh SB, et al. Toxoplasma gondii cyclic GMP-dependent kinase: Chemotherapeutic targeting of an essential parasite protein kinase. Eukaryotic Cell 2002;1:317-28; Nare B, Allocco J, Liberator PA, Donald RGK. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine Toxoplasmosis: Gamma interferon is required for efficacy. Antimicrob Agents Chemother 2002;46:300-7]. Compound 1 inhibits the growth of several related protozoan parasites of the subphylum Apicomplexa. Native PKG activity has been partially purified by cGMP-affinity and MonoQ ion exchange chromatography from Plasmodium falciparum (PfPKG). Biochemical fractions enriched for a 98kDa protein detected using anti-PKG antisera, contain cGMP-induced protein kinase activity that is sensitive to inhibition by Compound 1. To enable a more thorough characterization of PfPKG we expressed a synthetic cDNA incorporating T. gondii codon preference (Pf(Tg)PKG) in T. gondii parasites. The protein kinase activity of purified recombinant Pf(Tg)PKG is stimulated by cGMP, with significant cooperativity as demonstrated by a Hill coefficient of 2. Both substrate preference and inhibition of Pf(Tg)PKG kinase activity by Compound 1 are similar to that seen with native PfPKG, as well as PKG enzymes from Eimeria spp. and T. gondii. We conclude that PfPKG has biochemical and pharmacological properties that are similar to previously characterized apicomplexan PKG enzymes. Compound 1 is active against blood cell stages of P. falciparum cultured in vitro. In a Plasmodium berghei mouse model of infection, Compound 1 delays the onset of parasitemia but does not cure the parasite infection.

Potent 4-aminopiperidine based antimalarial agents.

A series of compounds with potent activity against a multi-drug-resistant strain of Plasmodium falciparum, the causative agent of the deadliest strain of malaria, is described. These compounds were also tested for cytotoxicity in human foreskin fibroblast assays, evaluated to determine their logD, and assayed for metabolism by human and murine hepatocytes. This work resulted in the development of compounds 9e and 10d, which showed good potency (IC(50)=75 nM and <60 nM, respectively, against Dd2), acceptable logD values, and reasonable metabolic stability.