An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus.

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.

Topical small molecule granzyme B inhibitor improves remodeling in a murine model of impaired burn wound healing.

Granzyme B (GzmB) is a serine protease that has long been thought to function exclusively in lymphocyte-mediated apoptosis. In recent years, this paradigm has been revisited due to the recognition that GzmB accumulates in the extracellular milieu in many autoimmune and chronic inflammatory disorders, and contributes to impaired tissue remodeling due to the cleavage of extracellular matrix proteins. Knockout studies suggest that GzmB-mediated cleavage of decorin (DCN) contributes to impaired collagen fibrillogenesis and remodeling. As DCN is anti-fibrotic and contributes to reduced hypertrophic scarring, GzmB-induced DCN cleavage could play a role in wound healing following burn injury. In the present study, a novel, gel-formulated, first-in-class small-molecule inhibitor of GzmB, VTI-1002, was assessed in a murine model of impaired, diabetic burn wound healing. VTI-1002 exhibited high specificity, potency, and target selectivity. Gel-formulated VTI-1002 was able to penetrate the stratum corneum and was retained in the skin with minimal systemic absorption. Daily topical administration of VTI-1002 gel for 30 days following thermal injury showed significantly accelerated wound closure, increased DCN protein levels, and collagen organization that was translated into significantly increased wound tensile strength compared to controls. Overall, VTI-1002 gel was well-tolerated in vivo and no adverse events were observed. Topical application of VTI-1002 represents a novel therapeutic approach for the treatment of cutaneous burn wounds.

Peptide-based inhibitors of the hepatitis C virus NS3 protease: structure-activity relationship at the C-terminal position.

The structure-activity relationship at the C-terminal position of peptide-based inhibitors of the hepatitis C virus NS3 protease is presented. The observation that the N-terminal cleavage product (DDIVPC-OH) of a substrate derived from the NS5A/5B cleavage site was a competitive inhibitor of the NS3 protease was previously described. The chemically unstable cysteine residue found at the P1 position of these peptide-based inhibitors could be replaced with a norvaline residue, at the expense of a substantial drop in the enzymatic activity. The fact that an aminocyclopropane carboxylic acid (ACCA) residue at the P1 position of a tetrapeptide such as 1 led to a significant gain in the inhibitory enzymatic activity, as compared to the corresponding norvaline derivative 2, prompted a systematic study of substituent effects on the three-membered ring. We report herein that the incorporation of a vinyl group with the proper configuration onto this small cycle produced inhibitors of the protease with much improved in vitro potency. The vinyl-ACCA is the first reported carboxylic acid containing a P1 residue that produced NS3 protease inhibitors that are significantly more active than inhibitors containing a cysteine at the same position.

NMR structural characterization of peptide inhibitors bound to the Hepatitis C virus NS3 protease: design of a new P2 substituent.

A comparative NMR conformational analysis of three distinct tetrapeptide inhibitors of the Hepatitis C NS3 protease that differ at the 4-aryloxy-substituted P2 proline position was undertaken. Specifically, transferred nuclear Overhauser effect experiments in combination with restrained systematic conformational searches were used to characterize the orientation of the P2 aryl substituents of these inhibitors when bound to the NS3 protease. Differences between free and bound conformations were also investigated. Analysis of the results allowed the design of a new P2 aromatic substituent, which significantly increased the potency of our inhibitors. The bound conformation of a specific competitive inhibitor having this novel P2 substituent is also described, along with a model of this inhibitor bound to the NS3 protease. This NS3 protease/inhibitor complex model also supports a hypothetical stabilization role for the P2 residue of the substrates and/or inhibitors and further elucidates the subtle details of the binding of the P2 residue of substrate-based inhibitors.

The Conformation of a Peptidyl Methyl Ketone Inhibitor Bound to the Human Cytomegalovirus Protease.

A weak inhibitor means faster exchange! Since the methyl ketone MK2 is a weak noncovalent peptidyl inhibitor of the human cytomegalovirus protease, exchange between the free and enzyme-bound forms is rapid. This allows for the use of transferred NOE NMR methods and molecular modeling, which show that the bound conformation of MK2 is an extended peptide. This is confirmed by the results of an X-ray crystallographic analysis of a related enzyme-inhibitor complex.

Novel antimicrobial lipopeptides with long in vivo half-lives.

The pharmacokinetic (PK) properties of novel lipopeptides (semi-synthetic amphomycin analogues) with potent activity against Gram-positive organisms were evaluated in mice and rats following single intravenous (i.v.) and oral administration. Following oral administration at 50mg/kg, plasma concentrations of amphomycin analogues were <0.3-0.9microg/mL, suggesting that oral availability was low. Following i.v. administration (5-10mg/kg), the majority of lipopeptides demonstrated a long half-life (5.2-8.0h in mice and 4.6-7.1h in rats), low clearance (0.005-0.016mL/min in mice and 0.050-0.084mL/min in rats) and a volume of distribution indicative of extracellular penetration (0.118-0.339L/kg in mice and 0.121-0.133L/kg in rats). The area under the plasma concentration-time curve extrapolated to infinity (AUC(0-infinity)) for a 10mg/kg i.v. dose was determined to be 601.7-791.7microgh/mL in mice and 511.1-850.2microgh/mL in rats. The long half-life and low clearance observed with these novel lipopeptides indicate that drug serum concentrations will remain above the target minimal inhibitory concentration (MIC) levels for significant periods of time. When combined with the potent efficacy of these agents against Gram-positive organisms, the results of the present study support further development of these lipopeptide analogues towards clinical evaluation.

Semisynthetic approaches to laspartomycin analogues.

Laspartomycin C (1), a lipopeptide antibiotic related to amphomycin, consists of a cyclic peptide core and an aspartic acid unit external to the core and linking this to a C15-2,3-unsaturated fatty acid. This was reported initially to be active against Staphylococcus aureus, and more recent studies have shown that it is active against VRE, VISA, and MRSA isolates. The enzymatic cleavage of the fatty acid tail was accomplished with a deacylase produced by Actinoplanes utahensis and resulted in two peptides, designated Peptide 1 and Peptide 2. Semisynthetic derivatives of both peptides have been made, and the principal requirement for biological activity appears to be the presence of an acylaspartic acid.

Optimization and determination of the absolute configuration of a series of potent inhibitors of human papillomavirus type-11 E1-E2 protein-protein interaction: a combined medicinal chemistry, NMR and computational chemistry approach.

We have previously reported the discovery and initial SAR optimization of the first series of inhibitors of the human papillomavirus type-11 (HPV11) E1-E2 protein-protein interaction. These inhibitors featured an indandione system spiro-fused onto an all syn substituted tetrahydrofuran ring. In this paper, we report new SAR efforts which have led to the identification of the first low nanomolar inhibitor of the HPV11 E1-E2 protein-protein interaction. In addition, we report a combined NMR and computational chemistry approach which allowed the successful determination of the absolute stereochemistry of the active species originating from the initial racemic lead.

Potent inhibitors of the hepatitis C virus NS3 protease: design and synthesis of macrocyclic substrate-based beta-strand mimics.

The virally encoded NS3 protease is essential to the life cycle of the hepatitis C virus (HCV), an important human pathogen causing chronic hepatitis, cirrhosis of the liver, and hepatocellular carcinoma. The design and synthesis of 15-membered ring beta-strand mimics which are capable of inhibiting the interactions between the HCV NS3 protease enzyme and its polyprotein substrate will be described. The binding interactions between a macrocyclic ligand and the enzyme were explored by NMR and molecular dynamics, and a model of the ligand/enzyme complex was developed.

Crystal structure of the E2 transactivation domain of human papillomavirus type 11 bound to a protein interaction inhibitor.

Interaction between the E2 protein and E1 helicase of human papillomaviruses (HPVs) is essential for the initiation of viral DNA replication. We recently described a series of small molecules that bind to the N-terminal transactivation domain (TAD) of HPV type 11 E2 and inhibits its interaction with E1 in vitro and in cellular assays. Here we report the crystal structures of both the HPV11 TAD and of a complex between this domain and an inhibitor, at 2.5- and 2.4-A resolution, respectively. The HPV11 TAD structure is very similar to that of the analogous domain of HPV16. Inhibitor binding caused no significant alteration of the protein backbone, but movements of several amino acid side chains at the binding site, in particular those of Tyr-19, His-32, Leu-94, and Glu-100, resulted in the formation of a deep hydrophobic pocket that accommodates the indandione moiety of the inhibitor. Mutational analysis provides functional evidence for specific interactions between Tyr-19 and E1 and between His-32 and the inhibitor. A second inhibitor molecule is also present at the binding pocket. Although evidence is presented that this second molecule makes only weak interactions with the protein and is likely an artifact of crystallization, its presence defines additional regions of the binding pocket that could be exploited to design more potent inhibitors.