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. Using visible absorption spectroscopy, we were able to demonstrate that this new inhibitor series binds specifically and reversibly to the NC with a peculiar 2:1 stoichiometry. A fluorescence-polarization-based binding assay was also developed in order to monitor the inhibitory activities of this series of inhibitors. To better characterize the structural aspect of inhibitor binding onto NC, we performed NMR studies using unlabeled and (13)C,(15)N-double-labeled NC(1-55) protein constructs. This allowed the determination of the solution structure of a ternary complex characterized by two inhibitor molecules binding to the two zinc knuckles of the NC protein. To the best of our knowledge, this represents the first report of a high-resolution structure of a small-molecule inhibitor bound to NC, demonstrating sub-micromolar potency and moderate antiviral potency with one analogue of the series. This structure was compared with available NC/oligonucleotide complex structures and further underlined the high flexibility of the NC protein, allowing it to adopt many conformations in order to bind its different oligonucleotide/nucleomimetic targets. In addition, analysis of the interaction details between the inhibitor molecules and NC demonstrated how this novel inhibitor series is mimicking the guanosine nucleobases found in many reported complex structures.

HTS promiscuity analyses for accelerating decision making.

Frequent hitters are compounds that are detected as a "hit" in multiple high-throughput screening (HTS) assays. Such behavior is specific (e.g., target family related) or unspecific (e.g., reactive compounds) or can result from a combination of such behaviors. Detecting such hits while predicting the underlying reason behind their promiscuous behavior is desirable because it provides valuable information not only about the compounds themselves but also about the assay methodology and target classes at hand. This information can also greatly reduce cost and time during HTS hit profiling. The present study exemplifies how to mine large HTS data repositories, such as the one at Boehringer Ingelheim, to identify frequent hitters, gain further insights into the causes of promiscuous behavior, and generate models for predicting promiscuous compounds. Applications of this approach are demonstrated using two recent large-scale HTS assays. The authors believe this analysis and its concrete applications are valuable tools for streamlining and accelerating decision-making processes during the course of hit discovery.

Development of specific "drug-like property" rules for carboxylate-containing oral drug candidates.

The carboxylate moiety is an important pharmacophore in the medicinal chemist's arsenal and is sometimes an irreplaceable functionality in drug-target interactions. Thus, practical guidance on its use in the most optimized manner would be a welcome addition to rational drug design. Key physicochemical and ADMET-PK properties from a dataset of drugs containing a carboxylate (COOH) moiety were assembled and compared with those of a broader, general drug dataset. Our main objective was to identify features specific to COOH-containing oral drugs that could be converted into simple rules delineating the boundaries within which prospective COOH-containing chemical series and COOH-containing drug candidates would be reasonably expected to possess properties suitable for oral administration. These specific "drug-like" property rules include molecular weight, the number of rotatable bonds, the number of hydrogen bond donors and acceptors, predictions of lipophilic character (calculated log P and log D values), topological polar surface area (TPSA), and the pK(a) value of the carboxylate moiety. Similar to the various sets of criteria that have emerged over the past decade and which have significantly reshaped the way medicinal chemists think about preferred drug chemical space, we propose these specific COOH "drug-like" property rules as a guide for the design of superior COOH-containing drug candidates and as a tool to better manage the liabilities generally associated with the presence of a COOH moiety.

Investigation on the role of the tetrazole in the binding of thiotetrazolylacetanilides with HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases.

The role of the tetrazole moiety in the binding of aryl thiotetrazolylacetanilides with HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases was explored. Different acyclic, cyclic and heterocyclic replacements were investigated in order to evaluate the conformational and electronic contribution of the tetrazole ring to the binding of the inhibitors in the NNRTI pocket. The replacement of the tetrazole by a pyrazolyl group led to reversal of selectivity, providing inhibitors with excellent potency against the double mutant reverse transcriptase.

Thiotetrazole alkynylacetanilides as potent and bioavailable non-nucleoside inhibitors of the HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases.

A series of aryl thiotetrazolylacetanilides were synthesized and found to be potent inhibitors of the HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases. The incorporation of an alkynyl fragment on the aniline provided inhibitors with excellent cellular activity and extensive SAR led to the identification of one inhibitor having good oral bioavailability in rats.

Scaffold hopping in the rational design of novel HIV-1 non-nucleoside reverse transcriptase inhibitors.

High-throughput screening hit 1 was identified as a potent, broad-spectrum, non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 replication. Analysis of the bound conformation of analogs of this inhibitor via molecular modeling and NMR contributed to the design of novel tertiary amide, carbamate, and thiocarbamate based NNRTIs.

Novel 8-substituted dipyridodiazepinone inhibitors with a broad-spectrum of activity against HIV-1 strains resistant to non-nucleoside reverse transcriptase inhibitors.

A series of novel 8-substituted dipyridodiazepinone-based inhibitors were investigated for their antiviral activity against wild type human immunodeficiency virus (HIV-1) and the clinically prevalent K103N/Y181C mutant virus. Our efforts have resulted in a series of benzoic acid analogues that are potent inhibitors of HIV-1 replication against a panel of HIV-1 strains resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Furthermore, the combination of good antiviral potency, a broad spectrum of activity, and an excellent pharmacokinetic profile provides strong justification for the further development of compound (7) as a potential treatment for wild type and NNRTI-resistant HIV-1 infection.

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.