The novel fungal CYP51 inhibitor VT-1598 displays classic dose-dependent antifungal activity in murine models of invasive aspergillosis.

Aspergillus spp. infections remain a global concern, with ∼30% attributable mortality of invasive aspergillosis (IA). VT-1598 is a novel fungal CYP51 inhibitor designed for exquisite selectivity versus human CYP enzymes to achieve a maximal therapeutic index and therefore maximal antifungal efficacy. Previously, its broad-spectrum in vitro antifungal activity was reported. We report here the pharmacokinetics (PK) and pharmacodynamics (PD) of VT-1598 in neutropenic mouse models of IA. The plasma area-under-the-curve (AUC) of VT-1598 increased nearly linearly between 5 and 40 mg/kg after 5 days of QD administration (155 and 1033 µg*h/ml, respectively), with a further increase with 40 mg/kg BID dosing (1354 µg*h/ml). When A. fumigatus isolates with in vitro susceptibilities of 0.25 and 1.0 µg/ml were used in a disseminated IA model, VT-1598 treatment produced no decrease in kidney fungal burden at QD 10 mg/kg, intermediate decreases at QD 20 mg/kg and maximum or near maximum decreases at 40 mg/kg QD and BID. The PK/PD relationships of AUCfree/MIC for 1-log killing for the two strains were 5.1 and 1.6 h, respectively, similar to values reported for approved CYP51 inhibitors. In a survival study where animals were observed for 12 days after the last treatment, survival was 100% at the doses tested (20 and 40 mg/kg QD), and fungal burden remained suppressed even though drug wash-out was complete. Similar dose-dependent reductions in lung fungal burden were observed in a pulmonary model of IA. These data strongly support further exploration of VT-1598 for the treatment of this lethal mold infection.

The novel fungal CYP51 inhibitor VT-1598 is efficacious alone and in combination with liposomal amphotericin B in a murine model of cryptococcal meningitis.

Objectives: Annual global deaths from cryptococcal meningitis (CM) are estimated at 180 000 and mortality is as high as 30%, even with optimal therapy. VT-1598 is a novel fungal CYP51 inhibitor with potent intrinsic antifungal activity against Cryptococcus. We report here VT-1598's in vivo antifungal activity in a murine model of CM. Methods: Single-dose plasma and brain pharmacokinetics in mice and MIC for Cryptococcus neoformans H99 were determined prior to efficacy studies. Short-course monotherapy and combination doses were explored with the endpoint of brain fungal burden. A survival study was also conducted using monotherapy treatment with fungal burden measured after a 6 day drug washout. Results: Oral doses of VT-1598 had good plasma and brain exposure and resulted in significant (P < 0.0001) and dose-dependent reductions in brain fungal burden, reaching a 6 log10 reduction. Unlike either positive drug control (fluconazole or liposomal amphotericin B), both mid and high doses of VT-1598 reduced fungal burden to below levels measured at the start of treatment. When VT-1598 was dosed in the survival study, no VT-1598-treated animal succumbed to the infection. Whereas fluconazole showed a 2.5 log10 increase in fungal burden after the 6 day washout, the VT-1598 mid- and high-dose animals showed almost no regrowth (<0.5 log10). In a separate fungal burden study using suboptimal doses of VT-1598 and liposomal amphotericin B to probe for combination effects, each combination had a positive effect relative to corresponding monotherapies. Conclusions: These pre-clinical in vivo data strongly support clinical investigation of VT-1598 as a novel therapy for this lethal infection.

Fungal CYP51 Inhibitors VT-1161 and VT-1129 Exhibit Strong In Vitro Activity against Candida glabrata and C. krusei Isolates Clinically Resistant to Azole and Echinocandin Antifungal Compounds.

The in vitro activities of fungal CYP51 inhibitors VT-1161 and VT-1129 were determined for Candida glabrata (n = 34) and C. krusei (n = 50). C. glabrata isolates were screened for FKS gene mutations. All isolates were resistant clinically and/or in vitro to at least one standard antifungal compound. VT-1161 and VT-1129 MICs for all isolates were at least 5-fold below achievable human plasma levels for VT-1161. VT-1161 and VT-1129 are promising for the treatment of resistant C. glabrata and C. krusei infections.

Efficacy of the clinical agent VT-1161 against fluconazole-sensitive and -resistant Candida albicans in a murine model of vaginal candidiasis.

Vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC) remain major health problems for women. VT-1161, a novel fungal CYP51 inhibitor which has potent antifungal activity against fluconazole-sensitive Candida albicans, retained its in vitro potency (MIC50 of ≤0.015 and MIC90 of 0.12 µg/ml) against 10 clinical isolates from VVC or RVVC patients resistant to fluconazole (MIC50 of 8 and MIC90 of 64 µg/ml). VT-1161 pharmacokinetics in mice displayed a high volume of distribution (1.4 liters/kg), high oral absorption (73%), and a long half-life (>48 h) and showed rapid penetration into vaginal tissue. In a murine model of vaginal candidiasis using fluconazole-sensitive yeast, oral doses as low as 4 mg/kg VT-1161 significantly reduced the fungal burden 1 and 4 days posttreatment (P < 0.0001). Similar VT-1161 efficacy was measured when an isolate highly resistant to fluconazole (MIC of 64 µg/ml) but fully sensitive in vitro to VT-1161 was used. When an isolate partially sensitive to VT-1161 (MIC of 0.12 µg/ml) and moderately resistant to fluconazole (MIC of 8 µg/ml) was used, VT-1161 remained efficacious, whereas fluconazole was efficacious on day 1 but did not sustain efficacy 4 days posttreatment. Both agents were inactive in treating an infection with an isolate that demonstrated weaker potency (MICs of 2 and 64 µg/ml for VT-1161 and fluconazole, respectively). Finally, the plasma concentrations of free VT-1161 were predictive of efficacy when in excess of the in vitro MIC values. These data support the clinical development of VT-1161 as a potentially more efficacious treatment for VVC and RVVC.

VT-1161 dosed once daily or once weekly exhibits potent efficacy in treatment of dermatophytosis in a guinea pig model.

Current therapies used to treat dermatophytoses such as onychomycosis are effective but display room for improvement in efficacy, safety, and convenience of dosing. We report here that the investigational agent VT-1161 displays potent in vitro antifungal activity against dermatophytes, with MIC values in the range of ≤0.016 to 0.5 µg/ml. In pharmacokinetic studies supporting testing in a guinea pig model of dermatophytosis, VT-1161 plasma concentrations following single oral doses were dose proportional and persisted at or above the MIC values for at least 48 h, indicating potential in vivo efficacy with once-daily and possibly once-weekly dosing. Subsequently, in a guinea pig dermatophytosis model utilizing Trichophyton mentagrophytes and at oral doses of 5, 10, or 25 mg/kg of body weight once daily or 70 mg/kg once weekly, VT-1161 was statistically superior to untreated controls in fungal burden reduction (P < 0.001) and improvement in clinical scores (P < 0.001). The efficacy profile of VT-1161 was equivalent to those for doses and regimens of itraconazole and terbinafine except that VT-1161 was superior to itraconazole when each drug was dosed once weekly (P < 0.05). VT-1161 was distributed into skin and hair, with plasma and tissue concentrations in all treatment and regimen groups ranging from 0.8 to 40 µg/ml (or µg/g), at or above the MIC against the isolate used in the model (0.5 µg/ml). These data strongly support the clinical development of VT-1161 for the oral treatment of onychomycosis using either once-daily or once-weekly dosing regimens.

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 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 [Kd], ≤ 39 nM) and similar to that of the pharmaceutical azole antifungals (Kd, ≤ 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.

Unexploited viral and host targets for the treatment of human immunodeficiency virus type 1 infection.

To date, all approved drugs for the treatment of infection by human immunodeficiency virus type 1 (HIV-1) target either of two viral enzymes, reverse transcriptase or protease. Drugs targeting different macromolecules could improve upon current shortcomings (ex, drug resistance, metabolism, toxicity, formulation) and provide foundations for novel combination therapies. This review will focus on the two key challenges for any new target--target validation (demonstrating the role in the disease), and target tractability (the likelihood of identifying modulators of that target that have drug-like properties). For this discussion, drug-like molecules are orally active, relatively small organic molecules. All of the virally-encoded proteins (other than reverse transcriptase and protease) and the host targets that have been postulated to be critical for HIV-1 proliferation will be reviewed.

A specific inhibitor of heart cytosolic 5'-nucleotidase I attenuates hydrolysis of adenosine 5'-monophosphate in primary rat myocytes.

ATP breakdown was triggered in primary rat myocytes in the presence of coformycin to force the catabolism of AMP through hydrolysis to adenosine. Selective inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium were used to measure the intracellular contribution of this enzyme to AMP hydrolysis under these conditions. The selective inhibitor 5-ethynyl-2',3'-dideoxyuridine inhibited the hydrolysis of AMP to adenosine in a concentration-dependent manner with an IC50 value of 20 microM. Maximal inhibition prevented 76% of the conversion of AMP to adenosine, indicating that under these conditions the majority of AMP hydrolysis in rat myocytes occurs through this enzyme. When ATP breakdown was triggered in the presence of thymidine 5'-phosphonate, a more potent inhibitor of the purified cytosolic 5'-nucleotidase, less inhibition of AMP hydrolysis occurred and only after prolonged preincubation of the myocytes with the inhibitor. These data demonstrate that the selective nucleoside inhibitors of c-N-I can effectively block the hydrolysis of AMP inside myocytes. Thus, these inhibitors may be useful tools in identifying the role of c-N-I during ATP catabolism in whole tissue and animal experiments.

N-Phenylamidines as selective inhibitors of human neuronal nitric oxide synthase: structure-activity studies and demonstration of in vivo activity.

Selective inhibition of the neuronal isoform of nitric oxide synthase (NOS) compared to the endothelial and inducible isoforms may be required for treatment of neurological disorders caused by excessive production of nitric oxide. Recently, we described N-(3-(aminomethyl)benzyl)acetamidine (13) as a slow, tight-binding inhibitor, highly selective for human inducible nitric oxide synthase (iNOS). Removal of a single methylene bridge between the amidine nitrogen and phenyl ring to give N-(3-(aminomethyl)phenyl)acetamidine (14) dramatically altered the selectivity to give a neuronal selective nitric oxide synthase (nNOS) inhibitor. Part of this large shift in selectivity was due to 14 being a rapidly reversible inhibitor of iNOS in contrast to the essentially irreversible inhibition of iNOS observed with 13. Structure-activity studies revealed that a basic amine functionality tethered to an aromatic ring and a sterically compact amidine are key pharmacophores for this class of NOS inhibitors. Maximal nNOS inhibition potency was achieved with N-(3-(aminomethyl)phenyl)-2-furanylamidine (77) (Ki-nNOS = 0.006 microM; Ki-eNOS = 0.35 microM; Ki-iNOS = 0.16 microM). Finally, alpha-fluoro-N-(3-(aminomethyl)phenyl)acetamidine (74) (Ki-nNOS = 0. 011 microM; Ki-eNOS = 1.1 microM; Ki-iNOS = 0.48 microM) had excellent brain penetration and inhibited nNOS in a rat brain slice assay as well as in the rat brain (cerebellum) in vivo. Thus, N-phenylamidines should be useful in validating the role of nNOS in neurological disorders.

Nucleotide and nucleoside analogues as inhibitors of cytosolic 5'-nucleotidase I from heart.

Substrate and product specificity studies were used to develop inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium. As measured by Vmax/Km, c-N-I preferred pyrimidine 2'-deoxyribonucleotides as substrates with thymidine monophosphate (TMP) being the most efficient. In product inhibition studies, thymidine inhibited noncompetitively and inorganic phosphate inhibited competitively, consistent with an ordered release of nucleoside prior to phosphate. Mirroring nucleotide substrate specificities, pyrimidine nucleosides were more potent product inhibitors than purine nucleosides. Thus, pyrimidine nucleotide and nucleoside analogues were developed as inhibitors. Phosphonate analogues of TMP were synthesized by a novel method. The most potent was the 5'-phosphonate of 3'-deoxythymidine (ddT) (apparent Ki value of 63 nM). In addition, pyrimidine nucleoside analogues were inhibitors with 5-ethynyl-2',3'-dideoxyuridine being the most potent (apparent Ki value of 3.7 microM). The most potent nucleotide and nucleoside inhibitor were both greater than 1000-fold more potent inhibiting c-N-I than the cytosolic 5'-nucleotidase II. The nucleoside analogue was also greater than 1000-fold more potent against c-N-I than the membrane ecto-5'-nucleotidase (e-N). Because the phosphonate analogues measurably inhibited e-N (apparent Ki values of 6-12 microM), the selectivity of the phosphonates for c-N-I versus e-N was less (40-200-fold). Because of the high selectivity for c-N-I versus both of the other 5'-nucleotidases, the nucleoside inhibitors of c-N-I may be useful biochemical tools in discerning the role that c-N-I plays in generating adenosine within myocardium.

Response of the septic vasculature to prolonged vasopressor therapy with N(omega)-monomethyl-L-arginine and epinephrine in canines.

OBJECTIVE: To investigate the effect of blocking nitric oxide production on cardiovascular function and survival in canine septic shock treated with or without a conventional vasopressor. DESIGN: Randomized, controlled trial. SETTING: An animal research laboratory at the National Institutes of Health. SUBJECTS: Sixty purpose-bred beagles. INTERVENTIONS: Fibrin clots containing Escherichia coli were surgically placed into the peritoneal cavity. N(omega)-monomethyl-L-arginine (L-NMMA) 10 mg/kg followed by 0.5, 1.0, or 4.0 mg/kg/hr), epinephrine (1 microg/kg/min), both, or neither were infused for 24 hrs beginning 6 hrs after the onset of infection. All animals received fluid and antibiotic therapy. MEASUREMENTS AND MAIN RESULTS: Serum nitric oxide metabolites, nitrite and nitrate, increased with infection (p = .024) and decreased with L-NMMA (p = .004, all doses combined). Myocardial nitric oxide synthase activity was ranked as follows: nonsurvivors > survivors > noninfected controls (p < .01). Other tissues examined showed the same pattern. L-NMMA produced sustained increases in systemic vascular resistance index and mean arterial pressure 9 and 24 hrs after the onset of infection (p < or = .04). Left ventricular ejection fraction was depressed by septic shock (p = .01) and further decreased by L-NMMA (p = .02). However, control and L-NMMA cardiac index values were similar (p > .4), perhaps because L-NMMA increased pulmonary artery occlusion pressure (p = .02). From 9 to 24 hrs, epinephrine, in the absence or presence of L-NMMA, blunted recovery of cardiac index (p < .02) and had a diminishing vasopressor effect (p = .05). Neither L-NMMA nor epinephrine, individually or combined, significantly altered survival rates at the doses investigated (p > or = .69). CONCLUSIONS: The tested doses showed that nitric oxide production was inhibited by L-NMMA in canine septic shock, but mortality and myocardial depression were unaffected. These results suggest that if L-NMMA has a beneficial effect on survival rates in septic shock, it is small.

Substituted N-phenylisothioureas: potent inhibitors of human nitric oxide synthase with neuronal isoform selectivity.

S-Ethyl N-phenylisothiourea (4) has been found to be a potent inhibitor of both the human constitutive and inducible isoforms of nitric oxide synthase. A series of substituted N-phenylisothiourea analogues was synthesized to investigate the structure-activity relationship of this class of inhibitor. Each analogue was evaluated for human isoform selectivity. One analogue, S-ethyl N-[4-(trifluoromethyl)phenyl]isothiourea (39), exhibited 115-fold and 29-fold selectivity for the neuronal isoform versus the inducible and endothelial derived constitutive isoforms, respectively. Studies have shown the substituted N-phenylisothiourea 39 binds competitively with L-arginine.

Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89.

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity.

1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo.

N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitric- oxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s-1 and a binding constant of 2.0 microM. Inhibition was dependent on the cofactor NADPH. L-Arginine was a competitive inhibitor of 1400W binding with a Ks value of 3.0 microM. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a Kd value

High-level expression of human inducible nitric oxide synthase in Chinese hamster ovary cells and characterization of the purified enzyme.

Chinese hamster ovary cells were transfected with the human inducible nitric oxide synthase (iNOS) cDNA under the control of the hCMV promoter. The NOS inhibitor, S-ethyl isothiourea, was utilized to minimize toxicity from NO production. Selected colonies were grown in the absence of S-ethyl isothiourea, after which nitrite levels in the medium were measured. NOS activities in cell lysates were determined, and 9 colonies had iNOS activities of over 1 nmol of citrulline formed/min/mg protein. iNOS expression was further increased by gene amplification and the use of sodium butyrate, resulting in two cell lines with stable activity of greater than 3 nmol/min/mg. The iNOS purified from these cells had a specific activity of over 500 nmol/min/mg, and its properties were similar to native iNOS purified from cytokine-induced DLD-1 cells. This is the most efficient expression system reported to date for iNOS.

Potent and selective inhibition of human nitric oxide synthases. Inhibition by non-amino acid isothioureas.

S-Ethylisothiourea was a potent competitive inhibitor of human nitric oxide synthase (NOS), with Ki values of 17, 36, and 29 nM for the inducible (i), endothelial (e), and neuronal (n) isozymes, respectively. Unlike some potent inhibitors of NOS, no time dependence was observed. S-Ethylisothiourea was not a detectable substrate for eNOS. S-Ethylisothiourea was also a potent inhibitor of mouse iNOS (Ki value of 5.2 nM), and its binding perturbed the spectrum of iNOS consistent with its altering the environment of the bound heme. The optimum binding of S-ethyl- and S-isopropylisothiourea relative to 70 other analogs suggested that these alkyl substitutions fit into a small hydrophobic pocket. Most isothioureas were 2-6-fold selective for the human iNOS (Ki for iNOS versus Ki for eNOS), with one being 19-fold selective. The cyclized mimics of S-ethylisothiourea, 2-NH2-thiazoline, and 2-NH2-thiazole, were also competitive inhibitors of human NOS. A third structural class of inhibitors, bisisothioureas, were, in general, the most selective in their inhibition of human iNOS. S,S'-(1,3-Phenylenebis(1,2-ethanediyl))bisisothiourea was 190-fold selective (Ki value of 0.047 microM against iNOS versus 9.0 microM against eNOS). These results demonstrate that potent and selective inhibition of human NOS isozymes is achievable.

Potent and selective inhibition of human nitric oxide synthases. Selective inhibition of neuronal nitric oxide synthase by S-methyl-L-thiocitrulline and S-ethyl-L-thiocitrulline.

Potent and selective inhibition of neuronal nitric oxide synthase (nNOS) compared to endothelial NOS (eNOS) and inducible NOS (iNOS) may be useful to treat cerebral ischemia (stroke) and other neurodegenerative diseases. S-Methyl-L-thiocitrulline (Me-TC) and S-ethyl-L-thiocitrulline (Et-TC) inhibited the oxidation of L-arginine and the L-arginine-independent oxidation of NADPH by nNOS from human brain. Me-TC and Et-TC were slow, tight binding inhibitors of nNOS with second-order association rate constants (kon) of 2.6 x 10(5) M-1 s-1 and 1.3 x 10(5) M-1 s-1, respectively. The respective dissociation rate constants (koff) were 3 x 10(-4) s-1 and 0.7 x 10(-4) s-1. Thus, the Kd values calculated from koff/kon were 1.2 and 0.5 nM, respectively. L-Arginine was a competitive inhibitor of Me-TC and Et-TC binding with competition constant (Ks) values of 2.2 and 2.7 microM, respectively. The Km of nNOS for L-arginine was 1.6 microM. The active site concentration of nNOS was estimated by titration with Et-TC. Based on this active site concentration, a kcat of 0.4 s-1 for the oxidation of L-arginine, was calculated. Me-TC and Et-TC were less potent inhibitors of human iNOS (Ki values of 34 and 17 nM, respectively) and human eNOS (Ki values of 11 and 24 nM). Thus, Me-TC and Et-TC were 10- and 50-fold, respectively, more potent inhibitors of nNOS than eNOS. Furthermore, Me-TC was also 17-fold selective for rat nNOS in neuronal tissue compared to rat eNOS in vascular endothelium, suggesting that Me-TC may be selective for nNOS in vivo and therefore, may be therapeutically useful to treat neurodegenerative diseases.