An assay recapitulating the 3′ processing activity of HIV-1 integrase (IN) was used to screen the Boehringer Ingelheim compound collection. Hit-to-lead and lead optimization beginning with compound 1 established the importance of the C3 and C4 substituent to antiviral potency against viruses with different aa124/ aa125 variants of IN. The importance of the C7 position on the serum shifted potency was established. Introduction of a quinoline substituent at the C4 position provided a balance of potency and metabolic stability. Combination of these findings ultimately led to the discovery of compound 26 (BI 224436), the first NCINI to advance into a phase Ia clinical trial.

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.

BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3=-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (

The development of peptidomimetic inhibitors of the human cytomegalovirus (HCMV) protease showing sub-micromolar potency in an enzymatic assay is described. Selective substitution of the amino acid residues of these inhibitors led to the identification of tripeptide inhibitors showing improvements in inhibitor potency of 27-fold relative to inhibitor 39 based upon the natural tetrapeptide sequence. Small side chains at P1 were well tolerated by this enzyme, a fact consistent with previous observations. The S2 binding pocket of HCMV protease was very permissive, tolerating lipophilic and basic residues. The substitutions tried at P3 indicated that a small increase in inhibitor potency could be realized by the substitution of a tert-leucine residue for valine. Substitutions of the N-terminal capping group did not significantly affect inhibitor potency. Pentafluoroethyl ketones, alpha,alpha-difluoro-beta-keto amides, phosphonates and alpha-keto amides were all effective substitutions for the activated carbonyl component and gave inhibitors which were selective for HCMV protease. A slight increase in potency was observed by lengthening the P1' residue of the alpha-keto amide series of inhibitors. This position also tolerated a variety of groups making this a potential site for future modifications which could modulate the physicochemical properties of these molecules.

Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptide backbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on the natural substrates and covering the P4 to P1' positions) has been determined at 2.7 A resolution. The inhibitor is bound in an extended conformation, forming an anti-parallel beta-sheet with the protease. The P3 and P1 side chains are less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding mode shows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serine proteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. In addition, large conformational differences relative to the structure of the free enzyme are observed, which may be important for inhibitor binding.