Conformation-based restrictions and scaffold replacements in the design of hepatitis C virus polymerase inhibitors: discovery of deleobuvir (BI 207127).

Conformational restrictions of flexible torsion angles were used to guide the identification of new chemotypes of HCV NS5B inhibitors. Sites for rigidification were based on an acquired conformational understanding of compound binding requirements and the roles of substituents in the free and bound states. Chemical bioisosteres of amide bonds were explored to improve cell-based potency. Examples are shown, including the design concept that led to the discovery of the phase III clinical candidate deleobuvir (BI 207127). The structure-based strategies employed have general utility in drug design.

Anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors with sub-micromolar potency in the cell-based replicon assay.

Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC50) and cell culture (EC50) potencies of less than 100 nanomolar.

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 2: structure-activity relationships (SAR) of the C3-phenyl moiety.

Detailed structure-activity relationships of the C3-phenyl moiety that allow for the optimization of antiviral potency of a series of 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione inhibitors of HIV capsid (CA) assembly are described. Combination of favorable substitutions gave additive SAR and allowed for the identification of the most potent compound in the series, analog 27. Productive SAR also transferred to the benzotriazepine and spirobenzodiazepine scaffolds, providing a solution to the labile stereocenter at the C3 position. The molecular basis of how compound 27 inhibits mature CA assembly is rationalized using high-resolution structural information. Our understanding of how compound 27 may inhibit immature Gag assembly is also discussed.

Discovery of a novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors.

A novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors were derived from a fragment-based approach using information from X-ray crystallographic analysis of NS5B-inhibitor complexes and iterative rounds of parallel synthesis. Structure-based drug design strategies led to the discovery of potent sub-micromolar inhibitors 11a-c and 12a-c from a weak-binding fragment-like structure 1 as a starting point.

Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography.

The HIV-1 capsid (CA) protein, a domain of Gag, which participates in formation of both the mature and immature capsid, represents a potential target for anti-viral drug development. Characterization of hits obtained via high-throughput screening of an in vitro capsid assembly assay led to multiple compounds having this potential. We previously presented the characterization of two inhibitor series that bind the N-terminal domain of the capsid (CA(NTD)), at a site located at the bottom of its helical bundle, often referred to as the CAP-1 binding site. In this work we characterize a novel series of benzimidazole hits. Initial optimization of this series led to compounds with improved in vitro assembly and anti-viral activity. Using NMR spectroscopy we found that this series binds to a unique site on CA(NTD), located at the apex of the helical bundle, well removed from previously characterized binding sites for CA inhibitors. 2D (1)H-(15)N HSQC and (19)F NMR showed that binding of the benzimidazoles to this distinct site does not affect the binding of either cyclophilin A (CypA) to the CypA-binding loop or a benzodiazepine-based CA assembly inhibitor to the CAP-1 site. Unfortunately, while compounds of this series achieved promising in vitro assembly and anti-viral effects, they also were found to be quite sensitive to a number of naturally occurring CA(NTD) polymorphisms observed among clinical isolates. Despite the negative impact of this finding for drug development, the discovery of multiple inhibitor binding sites on CA(NTD) shows that capsid assembly is much more complex than previously realized.

Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein.

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.

Implementation of an electronic medical record in family practice: a case study.

BACKGROUND: Electronic medical records (EMRs) have the potential to foster a safer, more effective and more efficient healthcare system. However, their implementation in primary care practice remains a challenge. OBJECTIVE: This study aims at exploring factors that have influenced the successful implementation of an EMR system in a family medicine group (FMG) in the Province of Québec, Canada. METHODS: A case study approach was selected to get a deep understanding of the phenomenon in its context. The case was chosen on the basis that it was the first FMG in Québec to implement a full EMR used by all clinicians. Fifteen semi-structured interviews were conducted with key informants. RESULTS: Factors that have influenced the success of the EMR implementation were classified under three broad themes: a project leader who combined the roles of clinical, technology and knowledge champion; an organisation that was open to and supportive of change; and an evidence-based implementation strategy tailored to the local context and adoption pace. CONCLUSIONS: This study underscores the importance of a champion for successful EMR implementation. It proposes a set of roles and characteristics that could be found in a champion as well as other elements for a successful EMR implementation strategy.

Biphenylsulfonacetic acid inhibitors of the human papillomavirus type 6 E1 helicase inhibit ATP hydrolysis by an allosteric mechanism involving tyrosine 486.

Human papillomaviruses (HPVs) are the causative agents of benign and malignant lesions of the epithelium. Despite their high prevalence, there is currently no antiviral drug for the treatment of HPV-induced lesions. The ATPase and helicase activities of the highly conserved E1 protein of HPV are essential for viral DNA replication and pathogenesis and hence are considered valid antiviral targets. We recently described novel biphenylsulfonacetic acid inhibitors of the ATPase activity of E1 from HPV type 6 (HPV6). Based on kinetics and mutagenesis studies, we now report that these compounds act by an allosteric mechanism. They are hyperbolic competitive inhibitors of the ATPase activity of HPV6 E1 and also inhibit its helicase activity. Compounds in this series can also inhibit the ATPase activity of the closely related enzyme from HPV11; however, the most potent inhibitors of HPV6 E1 are significantly less active against the type 11 protein. We identified a single critical residue in HPV6 E1, Tyr-486, substituted by a cysteine in HPV11, which is primarily responsible for this difference in inhibitor potency. Interestingly, HPV18 E1, which also has a tyrosine at this position, could be inhibited by biphenylsulfonacetic acid derivatives, thereby raising the possibility that this class of inhibitors could be optimized as antiviral agents against multiple HPV types. These studies implicate Tyr-486 as a key residue for inhibitor binding and define an allosteric pocket on HPV E1 that can be exploited for future drug discovery efforts.

Inhibitors of respiratory syncytial virus replication target cotranscriptional mRNA guanylylation by viral RNA-dependent RNA polymerase.

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in infants, immunocompromised patients, and the elderly. New antiviral agents would be important tools in the treatment of acute RSV disease. RSV encodes its own RNA-dependent RNA polymerase that is responsible for the synthesis of both genomic RNA and subgenomic mRNAs. The viral polymerase also cotranscriptionally caps and polyadenylates the RSV mRNAs at their 5' and 3' ends, respectively. We have previously reported the discovery of the first nonnucleoside transcriptase inhibitor of RSV polymerase through high-throughput screening. Here we report the design of inhibitors that have improved potency both in vitro and in antiviral assays and that also exhibit activity in a mouse model of RSV infection. We have isolated virus with reduced susceptibility to this class of inhibitors. The mutations conferring resistance mapped to a novel motif within the RSV L gene, which encodes the catalytic subunit of RSV polymerase. This motif is distinct from the catalytic region of the L protein and bears some similarity to the nucleotide binding domain within nucleoside diphosphate kinases. These findings lead to the hypothesis that this class of inhibitors may block synthesis of RSV mRNAs by inhibiting guanylylation of viral transcripts. We show that short transcripts produced in the presence of inhibitor in vitro do not contain a 5' cap but, instead, are triphosphorylated, confirming this hypothesis. These inhibitors constitute useful tools for elucidating the molecular mechanism of RSV capping and represent valid leads for the development of novel anti-RSV therapeutics.

A systematic approach to the optimization of substrate-based inhibitors of the hepatitis C virus NS3 protease: discovery of potent and specific tripeptide inhibitors.

The inadequate efficacy and tolerability of current therapies for the infectious liver disease caused by the hepatitis C virus have warranted significant efforts in the development of new therapeutics. We have previously reported competitive peptide inhibitors of the NS3 serine protease based on the N-terminal cleavage products of peptide substrates. A detailed study of the interactions of these substrate-based inhibitors with the different subsites of the serine protease active site led to the discovery of novel residues that increased the affinity of the inhibitors. In this paper, we report the combination of the best binding residues in a tetrapeptide series that resulted in extremely potent inhibitors that bind exquisitely well to this enzyme. A substantial increase in potency was obtained with the simultaneous introduction of a 7-methoxy-2-phenyl-4-quinolinoxy moiety at the gamma-position of the P2 proline and a tert-leucine as a P3 residue. The increase in potency allowed for the further truncation and led to the identification of tripeptide inhibitors. Structure activity relationship studies on this inhibitor series led to the identification of carbamate-containing tripeptides that are able to inhibit replication of subgenomic HCV RNA in cell culture with potencies below 1 microM. This inhibitor series has the potential of becoming antiviral agents for the treatment of HCV infections.

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.

Discovery of small-molecule inhibitors of the ATPase activity of human papillomavirus E1 helicase.

The Boehringer Ingelheim compound collection was screened for inhibitors of the ATPase activity of human papillomavirus E1 helicase to develop antiviral agents that inhibit human papillomavirus (HPV) DNA replication. This screen led to the discovery of (biphenyl-4-sulfonyl)acetic acid 1, which inhibits the ATPase activity of HPV type 6 E1 helicase with a low micromolar IC(50) value. A hit-to-lead exercise rapidly converted 1 into a low nanomolar lead series.

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.