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.

Antiplatelet and antithrombotic activity of RWJ-53308, a novel orally active glycoprotein IIb/IIIa antagonist.

RWJ-53308 is a novel nonpeptide glycoprotein IIb/IIIa (GPIIb/IIIa) antagonist that inhibits fibrinogen binding to GPIIb/IIIa with an IC(50) of 0.4+/-0.3 nM. RWJ-53308 inhibits thrombin-induced platelet aggregation in human gel-filtered platelets (IC(50)=60+/-12 nM) and platelet aggregation in human platelet-rich plasma (PRP) in response to collagen, arachidonic acid, ADP, and SFLLRN-NH(2) (IC(50)=60+/-10, 150+/-30, 70+/-4, and 160+/-80 nM, respectively). The potency of RWJ-53308 in dog and guinea pig PRP is similar to human PRP. RWJ-53308 inhibits ex vivo collagen- and ADP-induced platelet aggregation in conscious dogs for up to 4 h following 0.3 mg/kg iv, and through 4 and 6 h following 1 and 3 mg/kg po. Oral bioavailability is 16+/-7%. RWJ-53308 reduces thrombus weight in a canine arteriovenous (AV) shunt model following intravenous (0.01-0.1 mg/kg) and oral (3 mg/kg) administration. In a guinea pig carotid artery pinch-injury model, RWJ-53308 completely suppresses thrombus-induced cyclic flow reductions (CFR) at 0.7 mg/kg iv. RWJ-53308 also blocks thrombus formation in photoactivation- and ferric chloride-induced models of thrombosis in guinea pigs at 0.3 and 1 mg/kg iv, respectively. In summary, RWJ-53308 is a potent orally active GPIIb/IIIa antagonist that may be useful for both acute and chronic treatment of arterial thrombotic disorders.

1,2,4-triazolo[3,4-a]pyridine as a novel, constrained template for fibrinogen receptor (GPIIb/IIIa) antagonists.

Conformationally constrained analogues of the GPIIb/IIIa antagonist elarofiban (RWJ-53308) have been synthesized and biologically evaluated. The 1,2,4-triazolo[3,4-a]pyridine scaffold provided potent antagonists with favorable pharmacodynamic and pharmacokinetic attributes in dogs. Compounds 12a and 13a exhibited enhancements in oral bioavailability, t(1/2), and ex vivo duration of action (inhibition of ADP-induced platelet aggregation) relative to elarofiban.

Tritiated photoactivatable analogs of the native human thrombin receptor (PAR-1) agonist peptide, SFLLRN-NH2.

Six photoactivatable analogs of the human thrombin receptor activating peptide (TRAP), SFLLRN-NH2, were synthesized by substituting the photoactive amino acid, p-benzoylphenylalanine (Bpa), into each position of the peptide sequence. Platelet aggregation assays indicated that the peptides with Bpa substitutions at positions 3 to 6 retained agonist activity. These peptides were prepared in tritiated form as potential thrombin receptor photoaffinity labels. The [3H]Bpa-containing analogs were constructed by resynthesizing the peptides with the amino acid, 4-benzoyl-2',5'-dibromophenylalanine (Br2Bpa), and subjecting the purified peptides to Pd-catalyzed tritiodebromination. The radiochemical yields for the reductive tritiation were < 2% for peptides with [3H]Bpa in the third and fourth positions, and between 7 and 16% for the peptides with substitutions at the fifth and sixth positions. The low yields were due to over-reduction of the Bpa carbonyl group and nonspecific degradation during reductive tritiation. This report describes the first use of Br2Bpa for the preparation of tritiated photoactivatable peptides.

Protease-activated receptor-2 (PAR-2): structure-function study of receptor activation by diverse peptides related to tethered-ligand epitopes.

Protease-activated receptor-2 (PAR-2) is a tethered-ligand, G-protein-coupled receptor that is activated by proteolytic cleavage or by small peptides derived from its cleaved N-terminal sequence, such as SLIGRL-NH2. To assess specific PAR activity, we developed an immortalized murine PAR-1 (-/-) cell line transfected with either human PAR-2 or PAR-1. A "directed" library of more than 100 PAR agonist peptide analogues was synthesized and evaluated for PAR-2 and PAR-1 activity to establish an in-depth structure-function profile for specific action on PAR-2. The most potent agonist peptides (EC50 = 2-4 microM) had Lys at position 6, Ala at position 4, and pFPhe at position 2; however, these also exhibited potent PAR-1 activity (EC50 = 0.05-0.35 microM). We identified SLIARK-NH2 and SL-Cha-ARL-NH2 as relatively potent, highly selective PAR-2 agonists with EC50 values of 4 microM. Position 1 did not tolerate basic, acidic, or large hydrophobic amino acids. N-Terminal capping by acetyl eliminated PAR-2 activity, although removal of the amino group reduced potency by just 4-fold. At position 2, substitution of Leu by Cha or Phe gave equivalent PAR-2 potency, but this modification also activated PAR-1, whereas Ala, Asp, Lys, or Gln abolished PAR-2 activity; at position 3, Ile and Cha were optimal, although various amino acids were tolerated; at position 4, Ala or Cha increased PAR-2 potency 2-fold, although Cha introduced PAR-1 activity; at position 5, Arg or Lys could be replaced successfully by large hydrophobic amino acids. These results with hexapeptide C-terminal amides that mimic the native PAR-2 ligand indicate structural modes for obtaining optimal PAR-2 activity, which could be useful for the design of PAR-2 antagonists.

The 2-chlorotrityl resin: a worthy addition to the medicinal chemist's toolbox.

The polystyrene-based 2-chlorotrityl resin was originally used in the synthesis of peptides using an Fmoc-amino acid/carboxyl-linked protocol. While traditionally employed to prepare a number of biologically active peptides, the resin has received increasing attention as a support for the synthesis of pseudopeptide and non-peptide molecules recently. This review focuses on 2-chlorotrityl resin-supported synthesis of small molecules that collectively display a broad range of biological activities.

Quality control of radical prostatectomy: a feasibility study.

The aim of this study was to assess whether the quality of the surgical act could be an important prognostic factor for patients undergoing radical prostatectomy. This study also aims to investigate whether the surgical quality can be assessed by any means. Questionnaires were collected from 23 different institutes including 232 radical prostatectomies (RPr) performed for T1T2 prostate cancer. Blood loss, duration of surgery, margin status, postoperative prostate specific antigen (PSA) and urinary incontinence were analysed and correlated with the yearly number of RPr performed. The mean values obtained for each parameter were very different in the various centres. The outcome in terms of tumour control and incontinence could not be related to a higher or lower number of RPr performed. Quality control of RPr is feasible on the basis of an analysis of a few parameters, such as surgical margins, postoperative PSA and incontinence, that might recognise urologists that perform better or poorer than a proposed average.

Potent, orally active GPIIb/IIIa antagonists containing a nipecotic acid subunit. Structure-activity studies leading to the discovery of RWJ-53308.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significantly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor.

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.

Photoactivatable peptides based on BMS-197525: a potent antagonist of the human thrombin receptor (PAR-1).

Photoactivatable analogs of the human thrombin receptor (PAR-1) antagonist, N-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2 (BMS-197525), were prepared with benzophenone substitutions in the N-terminal, Leu, or Arg position. The analogs retained antagonist activity (with reduced potency); the tritium-labeled isotopomers are potential photoaffinity labels for the receptor. C-Terminal extension of the analogs with ornithine(biotin) did not significantly alter antagonist potency.

Combinatorial chemistry techniques applied to nonpeptide integrin antagonists.

The integrins are cell surface receptors that recognize extracellular matrix adhesive proteins such as fibrinogen, fibronectin, vitronectin, and VCAM-1 (vascular cell adhesion molecule-1). Nonpeptide integrin antagonists designed after the adhesion recognition sequence RGD (Arg-Gly-Asp) not only have displayed efficacy as antithrombotic agents, but also have promise for the treatment of cancer and osteoporosis. Combinatorial organic syntheses of chemical mini-libraries have facilitated nonpeptide lead optimization of integrin antagonists with marked success. Although these accomplishments have been realized primarily for the discovery of orally active GPIIb/IIIa antagonist antithrombotics, vitronectin receptor (avb3) antagonist research has also benefited from such rapid synthesis. The purpose of this review is to report progress in combinatorial synthesis lead optimization by highlighting the drug design strategies and synthetic tactics that have led to improved integrin antagonists.

Thrombin receptor (PAR-1) antagonists. Heterocycle-based peptidomimetics of the SFLLR agonist motif.

The thrombin receptor (PAR-1) is activated by alpha-thrombin to stimulate various cell types, including platelets, through the tethered-ligand sequence SFLLRN. A series of azole-based carboxamides, designed after SFLLR, were synthesized and evaluated in vitro. The compounds inhibited platelet aggregation induced by SFLLRN-NH2 or alpha-thrombin, and blocked the binding of [3H]-S-(p-F-Phe)-Har-L-Har-KY-NH2 to a CHRF membrane preparation of PAR-1. Oxazole 30 bound to PAR-1 with an IC50 of 1.6 microM, and gave IC50 values of 25 microM and 6.6 microM against alpha-thrombin- and SFLLRN-NH2-induced platelet aggregation, respectively.

NOE-derived conformation of GRGDSP cell adhesion recognition site in the presence of SDS micelles and integrin receptor GPIIB/IIIA.

The tripeptide RGD is well known for its role in integrin receptor-mediated cell-cell surface adhesion. Here, NMR and transferred NOE studies have been done with the fibrinogen/fibronectin-derived hexapeptide GRGDSP in the presence of sodium dodecyl sulfate (SDS) and purified platelet glycoprotein integrin receptor GPIIb/IIIa. In the presence of SDS and absence of receptor, GRGDSP gives NOE-based distance geometry-generated structures characteristic of two "nested' beta-turns centered at RG and GD. In the presence of integrin GPIIb/IIIa, GRGDSP resonances are chemically shifted and broadened consistent with a dynamic equilibrium between free and receptor "bound' peptide. NOEs characteristic of the nested beta-turns are either absent or weaker indicating a significant conformational change in GRGDSP in the receptor bound state. GRGDSP appears to bind the receptor in a more extended backbone conformation which positions aspartic acid and arginine residues spatially close for potential electrostatic interactions.

RGD induces conformational transition in purified platelet integrin GPIIb/IIIa-SDS system yielding multiple binding states for fibrinogen gamma-chain C-terminal peptide.

Fibrinogen gamma-chain C-terminal peptide HHLG-GAKQAGDV (gamma 12) and alpha-chain peptide GRGDSP are known to inhibit fibrinogen-mediated platelet cell aggregation via competitive interactions with platelet integrin receptor GPIIb/IIIa. NMR studies of gamma 12 in the presence of purified GPIIb/IIIa in SDS/water solution have demonstrated the presence of two gamma 12 binding states, one of which is eliminated by GRGDSP (RGD) up to a RGD: gamma 12 ratio of 2:1. RGD: gamma 12 ratios greater than 2:1 produce multiple sets of gamma 12 NMR signals in TOCSY spectra. At a ratio of 4:1, two to four such resonance sets can be resolved for A405, Q407, A408, G409, D410 and V411 spin systems. The number of multiple resonances remains unchanged at ratios of 6:1 and 8:1. Addition of gamma 12 to reverse the ratio to 8:8 (1:1) has no apparent effect on the RGD-induced distribution. Results suggest that RGD irreversibly induces a conformational transition(s) in GPIIb/IIIa to produce multiple gamma 12 binding sites on the receptor.

Design and evaluation of nonpeptide fibrinogen gamma-chain based GPIIb/IIIa antagonists.

Two series of nonpeptide turn mimetics were designed by analysis of the solution NMR structure of the 385-411 sequence of the gamma-chain of fibrinogen. These compounds, based on the KQAGD (Lys-Gln-Ala-Gly-Asp, 406-410) sequence, were synthesized and studied in vitro. The most interesting compound from our study, RWJ 50042 (25), exhibits potent inhibition of fibrinogen binding to GPIIb/IIIa (IC50 = 0.009 microM), as well as thrombin- or collagen-induced platelet aggregation (IC50 = 0.76, 0.14 microM). Since the 400-411 sequence is required for gamma-chain bioactivity and is a unique recognition sequence among ligands for integrins, vis-a-vis other RGD (Arg-Gly-Asp)-presenting proteins, these turn mimetics may represent a new, selective approach to antagonism of the fibrinogen receptor.

P-fimbriae of Escherichia coli as carriers for gonadotropin releasing hormone: development of a recombinant contraceptive vaccine.

The demand for an effective and low cost means of fertility control of domestic animals has raised interest in the development of contraceptive vaccines. A promising candidate for a vaccine component is the brain peptide gonadotropin releasing hormone (GnRH), which plays a central role in the regulation of reproductive functions in vertebrates. Neutralization of GnRH by vaccine-induced antibodies is expected to prevent the reproductive activity in a wide range of species. A GnRH-protein conjugate was prepared by means of recombinant DNA technology. The oligonucleotides encoding GnRH were inserted in hypervariable regions of the subunit gene of P-fimbriae of Escherichia coli. Hybrid fimbriae encoded by the manipulated fimbrial gene clusters were expressed efficiently on the cell surface of Escherichia coli. Vaccination of female rats and bull calves with purified hybrid GnRH-fimbriae strongly affected the reproductive characteristics. We conclude that P-fimbriae represent a very attractive carrier system for application of GnRH in a new type of vaccine.