BLIP-II Is a Highly Potent Inhibitor of Klebsiella pneumoniae Carbapenemase (KPC-2)

β-Lactamase inhibitory protein II (BLIP-II) is a potent inhibitor of class A β-lactamases. KPC-2 is a class A β-lactamase that is capable of hydrolyzing carbapenems and has become a widespread source of resistance to these drugs for Gram-negative bacteria. Determination of association and dissociation rate constants for binding between BLIP-II and KPC-2 reveals a very tight interaction with a calculated (k_off/k_on) equilibrium dissociation constant of 76 fM (76 × 10⁻¹⁵ M).     

The Antibiotic Micrococcin Is a Potent Inhibitor of Growth and Protein Synthesis in the Malaria Parasite

The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite.     

A Novel Inhibitor of Gyrase B Is a Potent Drug Candidate for Treatment of Tuberculosis and Nontuberculosis Mycobacterial Infections

New drugs to treat drug-resistant tuberculosis are urgently needed. Extensively drug-resistant and probably the totally drug-resistant tuberculosis strains are resistant to fluoroquinolones like moxifloxacin, which target gyrase A, and most people infected with these strains die within a year. In this study, we found that a novel aminobenzimidazole, VXc-486, which targets gyrase B, potently inhibits multiple drug-sensitive isolates and drug-resistant isolates of Mycobacterium tuberculosis in vitro (MICs of 0.03 to 0.30 μg/ml and 0.08 to 5.48 μg/ml, respectively) and reduces mycobacterial burdens in lungs of infected mice in vivo. VXc-486 is active against drug-resistant isolates, has bactericidal activity, and kills intracellular and dormant M. tuberculosis bacteria in a low-oxygen environment. Furthermore, we found that VXc-486 inhibits the growth of multiple strains of Mycobacterium abscessus, Mycobacterium avium complex, and Mycobacterium kansasii (MICs of 0.1 to 2.0 μg/ml), as well as that of several strains of Nocardia spp. (MICs of 0.1 to 1.0 μg/ml). We made a direct comparison of the parent compound VXc-486 and a phosphate prodrug of VXc-486 and showed that the prodrug of VXc-486 had more potent killing of M. tuberculosis than did VXc-486 in vivo. In combination with other antimycobacterial drugs, the prodrug of VXc-486 sterilized M. tuberculosis infection when combined with rifapentine-pyrazinamide and bedaquiline-pyrazinamide in a relapse infection study in mice. Furthermore, the prodrug of VXc-486 appeared to perform at least as well as the gyrase A inhibitor moxifloxacin. These findings warrant further development of the prodrug of VXc-486 for the treatment of tuberculosis and nontuberculosis mycobacterial infections.     

The Glucose Transporter PfHT1 Is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir

Malaria and HIV infection are coendemic in a large portion of the world and remain a major cause of morbidity and mortality. Growing resistance of Plasmodium species to existing therapies has increased the need for new therapeutic approaches. The Plasmodium glucose transporter PfHT is known to be essential for parasite growth and survival. We have previously shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters. While the PI lopinavir is known to have antimalarial activity, the mechanism of action is unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of glucose as their primary source of energy, and decreasing the available concentration of glucose leads to parasite death. We identified the malarial glucose transporter PfHT as a target for inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis for the antimalarial effect of lopinavir and provides a direct target for novel drug design with utility beyond the HIV-infected population.     

The ATP Binding Cassette Transporter Gene CgCDR1 from Candida glabrata Is Involved in the Resistance of Clinical Isolates to Azole Antifungal Agents

The resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrata clinical isolates recovered from two separate AIDS patients. The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itraconazole and ketoconazole. Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was reduced in the azole-resistant isolates, enhanced drug efflux was considered as a possible resistance mechanism. The expression of multidrug efflux transporter genes was therefore examined in the azole-susceptible and azole-resistant yeast isolates. For this purpose, C. glabrata genes conferring resistance to azole antifungals were cloned in a Saccharomyces cerevisiae strain in which the ATP binding cassette (ABC) transporter gene PDR5 was deleted. Three different genes were recovered, and among them, only C. glabrata CDR1 (CgCDR1), a gene similar to the Candida albicans ABC transporter CDR genes, was upregulated by a factor of 5 to 8 in the azole-resistant isolates. A correlation between upregulation of this gene and azole resistance was thus established. The deletion of CgCDR1 in an azole-resistant C. glabrata clinical isolate rendered the resulting mutant (DSY1041) susceptible to azole derivatives as the azole-susceptible clinical parent, thus providing genetic evidence that a specific mechanism was involved in the azole resistance of a clinical isolate. When CgCDR1 obtained from an azole-susceptible isolate was reintroduced with the help of a centromeric vector in DSY1041, azole resistance was restored and thus suggested that a trans-acting mutation(s) could be made responsible for the increased expression of this ABC transporter gene in the azole-resistant strain. This study demonstrates for the first time the determinant role of an ABC transporter gene in the acquisition of resistance to azole antifungals by C. glabrata clinical isolates.     

The Clinical Candidate VT-1161 Is a Highly Potent Inhibitor of Candida albicans CYP51 but Fails To Bind the Human Enzyme

The binding and cytochrome {"type":"entrez-protein","attrs":{"text":"P45051","term_id":"1171910","term_text":"P45051"}}P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 (ERG11) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, characteristic of heme iron coordination. The binding affinity of VT-1161 for Candida albicans CYP51 was high (dissociation constant [K_d], ≤39 nM) and similar to that of the pharmaceutical azole antifungals (K_d, ≤50 nM). In stark contrast, VT-1161 at concentrations up to 86 μM did not perturb the spectrum of recombinant human CYP51, whereas all the pharmaceutical azoles bound to human CYP51. In reconstitution assays, VT-1161 inhibited Candida albicans CYP51 activity in a tight-binding fashion with a potency similar to that of the pharmaceutical azoles but failed to inhibit the human enzyme at the highest concentration tested (50 μM). In addition, VT-1161 (MIC = 0.002 μg ml⁻¹) had a more pronounced fungal sterol disruption profile (increased levels of methylated sterols and decreased levels of ergosterol) than the known CYP51 inhibitor voriconazole (MIC = 0.004 μg ml⁻¹). Furthermore, VT-1161 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. In summary, VT-1161 potently inhibited Candida albicans CYP51 and culture growth but did not inhibit human CYP51, demonstrating a >2,000-fold selectivity. This degree of potency and selectivity strongly supports the potential utility of VT-1161 in the treatment of Candida infections.     

Spiroindolone That Inhibits PfATPase4 Is a Potent,Cidal Inhibitor of Toxoplasma gondii Tachyzoites In Vitro and In Vivo

Here, we show that spiroindolone, an effective treatment for plasmodia, is also active against Toxoplasma gondii tachyzoites. In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC₅₀], 1μM) and not toxic to human cells at ≥10μM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P = 0.002), measured 3 days after the last dose. This inhibition of T. gondii tachyzoites in vitro and in vivo indicates that spiroindolone is a promising lead candidate for further medicine development.     

The LABCG2 Transporter from the Protozoan Parasite Leishmania Is Involved in Antimony Resistance

Treatment for leishmaniasis, which is caused by Leishmania protozoan parasites, currently relies on a reduced arsenal of drugs. However, the significant increase in the incidence of drug therapeutic failure and the growing resistance to first-line drugs like antimonials in some areas of Northern India and Nepal limit the control of this parasitic disease. Understanding the molecular mechanisms of resistance in Leishmania is now a matter of urgency to optimize drugs used and to identify novel drug targets to block or reverse resistant mechanisms. Some members of the family of ATP-binding cassette (ABC) transporters in Leishmania have been associated with drug resistance. In this study, we have focused our interest to characterize LABCG2''s involvement in drug resistance in Leishmania. Leishmania major parasites overexpressing the ABC protein transporter LABCG2 were generated in order to assess how LABCG2 is involved in drug resistance. Assays of susceptibility to different leishmanicidal agents were carried out. Analysis of the drug resistance profile revealed that Leishmania parasites overexpressing LABCG2 were resistant to antimony, as they demonstrated a reduced accumulation of Sb^III due to an increase in drug efflux. Additionally, LABCG2 was able to transport thiols in the presence of Sb^III. Biotinylation assays using parasites expressing LABCG2 fused with an N-terminal green fluorescent protein tag revealed that LABCG2 is partially localized in the plasma membrane; this supports data from previous studies which suggested that LABCG2 is localized in intracellular vesicles that fuse with the plasma membrane during exocytosis. In conclusion, Leishmania LABCG2 probably confers antimony resistance by sequestering metal-thiol conjugates within vesicles and through further exocytosis by means of the parasite''s flagellar pocket.     

MK-7009, a Potent and Selective Inhibitor of Hepatitis C Virus NS3/4A Protease

The administration of hepatitis C virus (HCV) NS3/4A protease inhibitors to patients with chronic HCV infections has demonstrated that they have dramatic antiviral effects and that compounds acting via this mechanism are likely to form a key component of future anti-HCV therapy. We report here on the preclinical profile of MK-7009, an inhibitor of genotype 1a and 1b proteases at subnanomolar concentrations with modestly shifted potency against genotype 2a and 2b proteases at low nanomolar concentrations. Potent activity was also observed in a cell-based HCV replicon assay in the presence of added human serum (50%). In multiple species evaluated in preclinical studies, the MK-7009 concentrations in the liver were maintained at a significant multiple of the cell-based replicon 50% effective concentration over 12 to 24 h following the administration of moderate oral doses (5 to 10 mg per kg of body weight). MK-7009 also had excellent selectivity against both a range of human proteases and a broad panel of pharmacologically relevant ion channels, receptors, and enzymes. On the basis of this favorable profile, MK-7009 was selected for clinical development and is currently being evaluated in controlled clinical trials with both healthy volunteers and HCV-infected patients.Chronic infection with hepatitis C virus (HCV) is a major worldwide epidemic, and there are estimates that approximately 130 million to 170 million individuals are infected (17, 51). HCV is a positive-strand RNA virus of the Flaviviridae family and replicates primarily in the liver. While disease progression is typically a slow process that occurs over many years, a significant fraction of patients ultimately develop serious liver disease, including cirrhosis and hepatocellular carcinoma (19). Because of the major advances that have been made in therapy for human immunodeficiency virus (HIV) infection, HCV is currently a leading cause of death in HIV-coinfected patients (42) and is also the most common indication for liver transplantation surgery (1).HCV shows significant genetic heterogeneity, with six separate genotypes and multiple subtypes having been characterized to date (46). The current standard-of-care therapy for HCV infection involves treatment with a combination of pegylated interferon and ribavirin (10, 26, 31). While the rates of a sustained virologic response (SVR; defined as a viral load below the limit of detection 6 months after the cessation of treatment) are high for genotype 2- and 3-infected patients treated with pegylated interferon and ribavirin (44), the SVR rates in the more prevalent genotype 1-infected population are much lower, constituting 40 to 50% of treated individuals after 48 weeks of therapy. Pegylated interferon and ribavirin therapy is also associated with a number of serious side effects, which limit the number of patients who may be treated (9).Multiple viral proteins essential for replication have been characterized (4, 27); and a clinical proof of concept has been demonstrated for small-molecule inhibitors that act against several of these, including NS3/4A protease (40, 48), NS5B polymerase (both active site and allosteric inhibitors) (11, 12, 13, 36), NS4A (37), and most recently, NS5A (33). Of these, NS3/4A protease inhibitors have progressed the furthest to date in terms of clinical evaluation and have been demonstrated to achieve highly significant reductions in HCV viral loads in patients (47). The first clinical proof of concept for an HCV direct antiviral inhibitor was shown for BILN-2061 (Fig. ​(Fig.1)1) (16, 23), a rapidly reversible, P1-P3-constrained macrocyclic compound, although its development was subsequently discontinued as a consequence of the cardiac histology seen in monkeys (41). The clinically most advanced inhibitors acting via NS3/4A inhibition, VX-950 (telaprevir) (18, 35) and SCH-503034 (boceprevir) (43), are both keto-amide compounds which covalently bind to the active-site serine of the protease in a slowly reversible manner. More recently, a number of compounds structurally related to BILN-2061, including ITMN-191 (8), TMC435350 (49), and {"type":"entrez-nucleotide","attrs":{"text":"BI201335","term_id":"14667307","term_text":"BI201335"}}BI201335 (28), have progressed to the early stages of clinical evaluation.Open in a separate windowFIG. 1.NS3/4A protease inhibitors.We have previously described an approach to inhibitors of NS3/4A protease at subnanomolar concentrations utilizing a P2-P4 macrocyclic constraint in place of the P1-P3 linker used in the other rapidly reversible potent inhibitors described to date (21). In this report, we describe the preclinical profile of a development compound, MK-7009, a potent and selective NS3/4A protease inhibitor derived from further optimization of the P2-P4 series of macrocycles.     

ABT-378, a Highly Potent Inhibitor of the Human Immunodeficiency Virus Protease

The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (K_i = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC₅₀], 0.006 to 0.017 μM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC₅₀, ≤0.06 μM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC₅₀ in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC₅₀ for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.     

In Vitro Antiviral Activity of AG7088, a Potent Inhibitor of Human Rhinovirus 3C Protease

AG7088 is a potent, irreversible inhibitor of human rhinovirus (HRV) 3C protease (inactivation rate constant (k(obs)/[I]) = 1,470,000 +/- 440,000 M(-1) s(-1) for HRV 14) that was discovered by protein structure-based drug design methodologies. In H1-HeLa and MRC-5 cell protection assays, AG7088 inhibited the replication of all HRV serotypes (48 of 48) tested with a mean 50% effective concentration (EC(50)) of 0.023 microM (range, 0.003 to 0.081 microM) and a mean EC(90) of 0.082 microM (range, 0.018 to 0.261 microM) as well as that of related picornaviruses including coxsackieviruses A21 and B3, enterovirus 70, and echovirus 11. No significant reductions in the antiviral activity of AG7088 were observed when assays were performed in the presence of alpha(1)-acid glycoprotein or mucin, proteins present in nasal secretions. The 50% cytotoxic concentration of AG7088 was >1,000 microM, yielding a therapeutic index of >12,346 to >333,333. In a single-cycle, time-of-addition assay, AG7088 demonstrated antiviral activity when added up to 6 h after infection. In contrast, a compound targeting viral attachment and/or uncoating was effective only when added at the initiation of virus infection. Direct inhibition of 3C proteolytic activity in infected cells treated with AG7088 was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled proteins, which showed a dose-dependent accumulation of viral precursor polyproteins and reduction of processed protein products. The broad spectrum of antiviral activity of AG7088, combined with its efficacy even when added late in the virus life cycle, highlights the advantages of 3C protease as a target and suggests that AG7088 will be a promising clinical candidate.     

Safety,Pharmacokinetics, and Pharmacodynamics of TD‐0714, a Novel Potent Neprilysin Inhibitor in Healthy Adult and Elderly Subjects

TD‐0714 is an orally active, potent, and selective inhibitor of human neprilysin (NEP) in development for the treatment of chronic heart failure. Oral administration of TD‐0714 in rats resulted in dose‐dependent and sustained increases in plasma cyclic guanosine monophosphate (cGMP) over 24 hours consistent with NEP target engagement. Randomized, double‐blind, placebo controlled, single ascending dose (50–600 mg TD‐0714) and multiple ascending dose (10–200 mg TD‐0714 q.d. for 14 days) studies were conducted in healthy volunteers. TD‐0714 was generally well‐tolerated and no serious adverse events or clinically significant effects on vital signs or electrocardiogram parameters were observed. TD‐0714 exhibited dose‐proportional pharmacokinetics (PKs) with high oral bioavailability, minimal accumulation after once daily dosing, and negligible renal elimination. Pharmacodynamic (PD) responses were observed at all dose levels studied, as reflected by statistically significant increases in plasma cGMP concentrations. The increases in cGMP were significantly above the baseline (~ 50–100%) on day 14 for the entire 24‐hour interval indicating that sustained cGMP elevations are achieved at steady‐state. Maximal steady‐state cGMP response was observed in plasma and urine at doses ≥ 50 mg. The TD‐0714 PK‐PD relationship and safety profile were similar in elderly vs. younger adult subjects. The TD‐0714 PK and PD profiles support further clinical development of TD‐0714 and suggest the potential for once‐daily administration and predictable exposure in patients with cardiorenal diseases regardless of their renal function.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

☑ Neprilysin inhibitors (NEPis) in combination with other pathway inhibitors offer therapeutic advantage in patients with heart failure.

• WHAT QUESTION DID THIS STUDY ADDRESS?

☑ The study evaluates the safety and tolerability of second generation NEPi as well as whether its pharmacokinetic‐pharmacodynamic (PK‐PD) profile supports further evaluation in patients with heart failure.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

☑ The study demonstrates that this novel NEPi is tolerable in healthy volunteers, including elderly subjects as well as elicits a robust PD response at steady‐state thereby justifying further evaluation and providing dose recommendations for phase II.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

☑ The study adds to the armamentarium of drugs with potential for significant improvement in heart failure outcomes. Through nontraditional methods of administering microtracer i.v. dose as well as PD assessments in both single ascending dose and multiple ascending dose portions of the study, the study exemplifies how phase I studies can be used efficiently to investigate PK properties of investigational drugs as well as provide clear PK‐PD‐based dose recommendations for future clinical studies.

Chronic heart failure (CHF) is a complex clinical syndrome that results from functional impairment of ventricular filling or ejection of blood, designated as heart failure with preserved or reduced ejection fraction (HFrEF), respectively. ¹ Progression of CHF may, in part, be due to inadequate compensation by protective endogenous neurohormonal systems, which include the natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide, and C‐type natriuretic peptide. ² Human neprilysin (hNEP) is the enzyme responsible, at least in part, for degradation of natriuretic peptides. ³ Inhibition of neprilysin (NEP), therefore, leads to elevations in endogenous natriuretic peptide levels, which exert protective cardiorenal effects, via a cyclic guanosine monophosphate (cGMP)‐dependent pathway, including vasodilation, diuresis/natriuresis, antiproliferative, antifibrotic, and antihypertrophic effects. ² , ⁴ Inhibition of NEP may provide additional cardioprotective effects in addition to inhibition of the renin angiotensin aldosterone system pathway. LCZ696, a combination of an NEP inhibitor (sacubitril) and an angiotensin II receptor blocker (valsartan), has been shown to be more effective than enalapril (angiotensin‐converting enzyme inhibitor), on top of standard of care (including beta‐blockers and diuretics) treatments for HFrEF. ⁵ TD‐0714 is an orally active, potent, and highly selective inhibitor of hNEP that is being developed as an investigational compound for the treatment of CHF. TD‐0714 is a potent competitive inhibitor of hNEP (K_i = 0.427 nM, dissociation terminal half‐life (t_1/2) determined from k_off = 144 minutes) ⁶ with high selectivity for hNEP over a range of other molecular targets, including human angiotensin‐converting enzyme (ACE) and human amyloid precursor protein (internal data). Selectivity over ACE and amyloid precursor protein is important to avoid the adverse effects of angioedema reported previously for ACE‐NEP inhibitors, such as omapatrilat. ⁷ In addition, TD‐0714 was > 10‐fold more potent compared to LBQ657, which is the active metabolite of sacubitril. The aim of the study described here was to evaluate the safety and pharmacokinetics‐pharmacodynamics (PKs‐PDs) of TD‐0714 to support further evaluation in patients with heart failure. Single ascending dose (SAD) and multiple ascending dose (MAD) studies in healthy volunteers demonstrate that TD‐0714 was well‐tolerated, including elderly subjects. Furthermore, TD‐0714 PK was well‐characterized and translated to significantly increased cGMP levels in plasma and urine indicating robust PD response supportive of once‐daily dosing.     

Remogliflozin Etabonate,a Selective Inhibitor of the Sodium-Glucose Transporter 2, Improves Serum Glucose Profiles in Type 1 Diabetes

OBJECTIVE

Remogliflozin etabonate (RE), an inhibitor of the sodium-glucose transporter 2, improves glucose profiles in type 2 diabetes. This study assessed safety, tolerability, pharmacokinetics, and pharmacodynamics of RE in subjects with type 1 diabetes.

RESEARCH DESIGN AND METHODS

Ten subjects managed with continuous subcutaneous insulin infusion were enrolled. In addition to basal insulin, subjects received five randomized treatments: placebo, prandial insulin, 50 mg RE, 150 mg RE, and mg RE 500.

RESULTS

Adverse events and incidence of hypoglycemia with RE did not differ from placebo and prandial insulin groups. RE significantly increased urine glucose excretion and reduced the rise in plasma glucose concentration after oral glucose. RE reduced incremental adjusted weighted mean glucose (0–4 h) values by 42–49 mg/dL and mean glucose (0–10 h) by 52–69 mg/dL.

CONCLUSIONS

RE can be safely administered with insulin in type 1 diabetes and reduces plasma glucose concentrations compared with placebo.Remogliflozin etabonate (RE) is an oral prodrug of remogliflozin (1), a selective antagonist of the sodium-dependent glucose transporter 2 (SGLT2) located in renal proximal tubules (2–4). It lowers glucose concentrations in type 2 diabetes by inhibiting renal glucose reabsorption (5). Because this mechanism functions independently of insulin, RE could be an effective oral adjunct to insulin for treatment of type 1 diabetes. This clinical trial evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of RE administered to subjects with type 1 diabetes. This is the first report of administration of an SGLT2 inhibitor in this patient population.