Clinical targeting of HIV capsid protein with a long-acting small molecule.

Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment-which contributes to virologic failure, resistance generation and viral transmission-as well as of pre-exposure prophylaxis, leading to new infections.

Discovery of Potent Cyclophilin Inhibitors Based on the Structural Simplification of Sanglifehrin A.

Cyclophilin inhibition has been a target for the treatment of hepatitis C and other diseases, but the generation of potent, drug-like molecules through chemical synthesis has been challenging. In this study, a set of macrocyclic cyclophilin inhibitors was synthesized based on the core structure of the natural product sanglifehrin A. Initial compound optimization identified the valine-m-tyrosine-piperazic acid tripeptide (Val-m-Tyr-Pip) in the sanglifehrin core, stereocenters at C14 and C15, and the hydroxyl group of the m-tyrosine (m-Tyr) residue as key contributors to compound potency.

Characterization of protein-protein interactions by isothermal titration calorimetry.

The analysis of protein-protein interactions has attracted the attention of many researchers from both a fundamental point of view and a practical point of view. From a fundamental point of view, the development of an understanding of the signaling events triggered by the interaction of two or more proteins provides key information to elucidate the functioning of many cell processes. From a practical point of view, understanding protein-protein interactions at a quantitative level provides the foundation for the development of antagonists or agonists of those interactions.

Discovery of GS-9669, a thumb site II non-nucleoside inhibitor of NS5B for the treatment of genotype 1 chronic hepatitis C infection.

Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure-activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding.

Large-scale functional purification of recombinant HIV-1 capsid.

During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents.

Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4.

Novel, potent and orally bioavailable indolizidinone-derived inhibitors of the hepatitis C virus NS3 protease.

A novel, potent, and orally bioavailable class of product-like inhibitors of the HCV NS3 protease was discovered by constraining the P2-P3 amide bond and the P3 hydrocarbon substituent to the protease-bound conformation. This preorganization was accomplished by incorporation of the P2-P3 amide into a six-membered ring attached to the P2-proline 5-position. Isothermal calorimetric characterization of the role of hydrocarbon substitution of this six-membered ring, upon binding the HCV NS3 protease, was found to be exclusively entropic in nature.

Structural and binding analysis of pyrimidinol carboxylic acid and N-hydroxy quinazolinedione HIV-1 RNase H inhibitors.

HIV-1 RNase H breaks down the intermediate RNA-DNA hybrids during reverse transcription, requiring two divalent metal ions for activity. Pyrimidinol carboxylic acid and N-hydroxy quinazolinedione inhibitors were designed to coordinate the two metal ions in the active site of RNase H. High-resolution (1.

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile.

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K(i) = 8.

The macroscopic rate of nucleic acid translocation by hepatitis C virus helicase NS3h is dependent on both sugar and base moieties.

The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3'-to-5' superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s(-1) (oligo(dT)), 35 nt s(-1) (oligo(dU)), and 42 nt s(-1) (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation.

RNase H active site inhibitors of human immunodeficiency virus type 1 reverse transcriptase: design, biochemical activity, and structural information.

Pyrimidinol carboxylic acids were designed as inhibitors of HIV-1 RNase H function. These molecules can coordinate to two divalent metal ions in the RNase H active site. Inhibition of enzymatic activity was measured in a biochemical assay, but no antiviral effect was observed.

Thermodynamic benchmark study using Biacore technology.

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants.

HIV-1 integrase preassembled on donor DNA is refractory to activity stimulation by LEDGF/p75.

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values.

Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology.

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively.

Suppression of HIV-1 protease inhibitor resistance by phosphonate-mediated solvent anchoring.

The introduction of human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) markedly improved the clinical outcome and control of HIV-1 infection. However, cross-resistance among PIs due to a wide spectrum of mutations in viral protease is a major factor limiting their broader clinical use. Here we report on the suppression of PI resistance using a covalent attachment of a phosphonic acid motif to a peptidomimetic inhibitor scaffold.

Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro.

SARS (severe acute respiratory syndrome) is caused by a newly discovered coronavirus. A key enzyme for the maturation of this virus and, therefore, a target for drug development is the main protease 3CL(pro) (also termed SARS-CoV 3CL(pro)). We have cloned and expressed in Escherichia coli the full-length SARS-CoV 3CL(pro) as well as a truncated form containing only the catalytic domains.

Characterization of protein-protein interactions by isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is a powerful technique to study both protein-ligand and protein-protein interactions. This methods chapter is devoted to describing protein-protein interactions, in particular, the association between two different proteins and the self-association of a protein into homodimers. ITC is the only technique that determines directly the thermodynamic parameters of a given reaction: DeltaG, DeltaH, DeltaS, and DeltaCP.

Interactions of HIV-1 proteins gp120 and Nef with cellular partners define a novel allosteric paradigm.

During the course of infection, a subset of HIV-1 proteins interacts with multiple cellular partners, sometimes in a hierarchical or sequential way. These proteins include those associated with the initial infection event, with the preparation of the cell for the replicative cycle of the virus and with the exit of new virions from the infected cell. It appears that the interactions of viral proteins with multiple cellular partners are mediated by the occurrence of ligand-induced conformational changes that direct the binding of these proteins to subsequent partners.

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites.

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding.

Beta-turn Phe in HIV-1 Env binding site of CD4 and CD4 mimetic miniprotein enhances Env binding affinity but is not required for activation of co-receptor/17b site.

HIV-1 enters a host cell after an initial interaction between viral envelope glycoprotein gp120 and cell surface receptor CD4, followed by a second interaction between gp120 and a cell surface chemokine receptor. CD4 residue Phe43 makes a significant contribution to the high-affinity interaction between CD4 and env. We and others have used scorpion toxin scaffolds to display and examine CD4 epitopes used for gp120 recognition.

The linkage between protein folding and functional cooperativity: two sides of the same coin?

During the course of their biological function, proteins undergo different types of structural rearrangements ranging from local to large-scale conformational changes. These changes are usually triggered by their interactions with small-molecular-weight ligands or other macromolecules. Because binding interactions occur at specific sites and involve only a small number of residues, a chain of cooperative interactions is necessary for the propagation of binding signals to distal locations within the protein structure.