Discovery of novel small-molecule HIV-1 replication inhibitors that stabilize capsid complexes.

The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex.

A novel inhibitor-binding site on the HIV-1 capsid N-terminal domain leads to improved crystallization via compound-mediated dimerization.

Despite truly impressive achievements in the global battle against HIV there remains a need for new drugs directed against novel targets, and the viral capsid protein (CA) may represent one such target. Intense structural characterization of CA over the last two decades has provided unprecedented insight into the structure and assembly of this key viral protein. Furthermore, several inhibitor-binding sites that elicit antiviral activity have been reported on CA, two of which are located on its N-terminal domain (CANTD).

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.

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 1: addressing configurational instability through scaffold modification.

The optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly that possess a labile stereocenter at C3 is described. Quaternization of the C3 position of compound 1 in order to prevent racemization gave compound 2, which was inactive in our capsid disassembly assay. A likely explanation for this finding was revealed by in silico analysis predicting a dramatic increase in energy of the bioactive conformation upon quaternization of the C3 position.

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.

Discovery and structural characterization of a new inhibitor series of HIV-1 nucleocapsid function: NMR solution structure determination of a ternary complex involving a 2:1 inhibitor/NC stoichiometry.

The nucleocapsid (NC) protein is an essential factor with multiple functions within the human immunodeficiency virus type 1 (HIV-1) replication cycle. In this study, we describe the discovery of a novel series of inhibitors that targets HIV-1 NC protein by blocking its interaction with nucleic acids. This series was identified using a previously described capsid (CA) assembly assay, employing a recombinant HIV-1 CA-NC protein and immobilized TG-rich deoxyoligonucleotides.

Monitoring binding of HIV-1 capsid assembly inhibitors using (19)F ligand-and (15)N protein-based NMR and X-ray crystallography: early hit validation of a benzodiazepine series.

The emergence of resistance to existing classes of antiretroviral drugs underlines the need to find novel human immunodeficiency virus (HIV)-1 targets for drug discovery. The viral capsid protein (CA) represents one such potential target. Recently, a series of benzodiazepine inhibitors was identified via high-throughput screening using an in vitro capsid assembly assay (CAA).

Inhibition of HIV-1 capsid assembly: optimization of the antiviral potency by site selective modifications at N1, C2 and C16 of a 5-(5-furan-2-yl-pyrazol-1-yl)-1H-benzimidazole scaffold.

A uHTS campaign led to the discovery of a 5-(5-furan-2-ylpyrazol-1-yl)-1H-benzimidazole series that inhibits assembly of HIV-1 capsid. Synthetic manipulations at N1, C2 and C16 positions improved the antiviral potency by a . The X-ray structure of 33 complexed with the capsid N-terminal domain allowed identification of major interactions between the inhibitor and the protein.

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.

Combined X-ray, NMR, and kinetic analyses reveal uncommon binding characteristics of the hepatitis C virus NS3-NS4A protease inhibitor BI 201335.

Hepatitis C virus infection, a major cause of liver disease worldwide, is curable, but currently approved therapies have suboptimal efficacy. Supplementing these therapies with direct-acting antiviral agents has the potential to considerably improve treatment prospects for hepatitis C virus-infected patients. The critical role played by the viral NS3 protease makes it an attractive target, and despite its shallow, solvent-exposed active site, several potent NS3 protease inhibitors are currently in the clinic.

Discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly.

The discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly is described. Synthesis of analogs of the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione hit established structure-activity relationships. Replacement of the enamine functionality of the hit series with either an imidazole or a pyrazole ring led to compounds that inhibited both capsid assembly and reverse transcriptase.

Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335).

C-Terminal carboxylic acid containing inhibitors of the NS3 protease are reported. A novel series of linear tripeptide inhibitors that are very potent and selective against the NS3 protease are described. A substantial contribution to the potency of these linear inhibitors arises from the introduction of a C8 substituent on the B-ring of the quinoline moiety found on the P2 of these inhibitors.

Small molecule inhibitors of the human papillomavirus E1-E2 interaction.

Human papillomaviruses are responsible for multiple human diseases, including cervical cancer caused by multiple high-risk types and genital warts caused by the low-risk types 6 and 11. Based on the research indicating that low-risk HPV could be successfully targeted by inhibitors of viral DNA replication, we carried out several high-throughput screens for inhibitors of DNA replication activities. Two series were identified in screens for inhibitors of the interaction between the viral proteins E1 and E2.

Use of the fused NS4A peptide-NS3 protease domain to study the importance of the helicase domain for protease inhibitor binding to hepatitis C virus NS3-NS4A.

The NS3 protein of hepatitis C virus is unusual because it encodes two unrelated enzymatic activities in linked protease and helicase domains. It has also been intensively studied because inhibitors targeting its protease domain have potential to significantly improve treatment options for those infected with this virus. Many enzymological studies and inhibitor discovery programs have been carried out using the isolated protease domain in complex with a peptide derived from NS4A which stimulates activity.

Potent triazolyl-proline-based inhibitors of HCV NS3 protease.

The design and synthesis of tripeptide-based inhibitors of the HCV NS3 protease containing a novel P2-triazole is described. Replacement of the P2 quinoline with a triazole moiety provided a versatile handle which could be expediently modified to generate a diverse series of inhibitors. Further refinement by the incorporation of an aryl-substituted triazole and replacement of the P1 acid with an acyl sulfonamide ultimately provided inhibitors with interesting cellular activity.

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.

Quantifying trifluoroacetic acid as a counterion in drug discovery by 19F NMR and capillary electrophoresis.

Drug discovery compounds are often isolated as salts of trifluoroacetate from preparative high performance liquid chromatography, which are then used for biological assays in order to assess their efficacy against the biochemical target of interest. It is, therefore, imperative to determine the TFA content in order to ascertain the correct formula weight and when required, to ensure that the TFA has been completely exchanged for another counterion in order to have superior pharmacokinetic properties and to avoid potential toxicity effects. In this paper, we present capillary electrophoresis and (19)F nuclear magnetic resonance methods for determining the TFA content of drug discovery compounds.

The therapeutic potential of NS3 protease inhibitors in HCV infection.

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide and afflicts > 170 million people. The HCV-encoded NS3 protease is essential for viral replication and has long been recognised as a prime target for antiviral drugs. However, the peculiar active site structure of this enzyme, a shallow dent on the surface of the protein, has rendered the development of small-molecule inhibitors a highly challenging task.

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.

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.

Novel azapeptide inhibitors of hepatitis C virus serine protease.

Azapeptides are known inhibitors of several serine and cysteine proteases. In seeking different classes of inhibitors for the HCV serine protease, a series of novel azapeptide-based inhibitors were investigated which incorporated noncleavable P1/P1' aza-amino acyl residues. Extensive SAR studies around the P1/P1' aza-amino acyl fragment resulted in the identification of potent and selective inhibitors.

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.

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.

Discovery of the first series of inhibitors of human papillomavirus type 11: inhibition of the assembly of the E1-E2-Origin DNA complex.

We have discovered a series of inhibitors of the assembly of the HPV11 E1-E2-origin DNA complex, which incorporate an indandione fused to a substituted tetrahydrofuran.