Milan Kožíšek

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.

Urea and guanidinium induced denaturation of a Trp-cage miniprotein.

Using a combination of experimental techniques (circular dichroism, differential scanning calorimetry, and NMR) and molecular dynamics simulations, we performed an extensive study of denaturation of the Trp-cage miniprotein by urea and guanidinium. The experiments, despite their different sensitivities to various aspects of the denaturation process, consistently point to simple, two-state unfolding process. Microsecond molecular dynamics simulations with a femtosecond time resolution allow us to unravel the detailed molecular mechanism of Trp-cage unfolding. The process starts with a destabilizing proline shift in the hydrophobic core of the miniprotein, followed by a gradual destruction of the hydrophobic loop and the α-helix. Despite differences in interactions of urea vs guanidinium with various peptide moieties, the overall destabilizing action of these two denaturants on Trp-cage is very similar.

Ninety-nine is not enough: molecular characterization of inhibitor-resistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region

ABSTRACT While the selection of amino acid insertions in human immunodeficiency virus (HIV) reverse transcriptase (RT) is a known mechanism of resistance against RT inhibitors, very few reports on the selection of insertions in the protease (PR) coding region have been published. It is still unclear whether these insertions impact protease inhibitor (PI) resistance and/or viral replication capacity. We show that the prevalence of insertions, especially between amino acids 30 to 41 of HIV type 1 (HIV-1) PR, has increased in recent years. We identified amino acid insertions at positions 33 and 35 of the PR of HIV-1-infected patients who had undergone prolonged treatment with PIs, and we characterized the contribution of these insertions to viral resistance. We prepared the corresponding mutated, recombinant PR variants with or without insertions at positions 33 and 35 and characterized them in terms of enzyme kinetics and crystal structures. We also engineered the corresponding recombinant viruses and analyzed the PR susceptibility and replication capacity by recombinant virus assay. Both in vitro methods confirmed that the amino acid insertions at positions 33 and 35 contribute to the viral resistance to most of the tested PIs. The structural analysis revealed local structural rearrangements in the flap region and in the substrate binding pockets. The enlargement of the PR substrate binding site together with impaired flap dynamics could account for the weaker inhibitor binding by the insertion mutants. Amino acid insertions in the vicinity of the binding cleft therefore represent a novel mechanism of HIV resistance development.

Structure-aided design of novel inhibitors of HIV protease based on a benzodiazepine scaffold.

HIV protease is a primary target for the design of virostatics. Screening of libraries of non-peptide low molecular weight compounds led to the identification of several new compounds that inhibit HIV PR in the low micromolar range. X-ray structure of the complex of one of them, a dibenzo[b,e][1,4]diazepinone derivative, showed that two molecules of the inhibitor bind to the PR active site. Covalent linkage of two molecules of such a compound by a two-carbon linker led to a decrease of the inhibition constant of the resulting compound by 3 orders of magnitude. Molecular modeling shows that these dimeric inhibitors form two crucial hydrogen bonds to the catalytic aspartates that are responsible for their improved activity compared to the monomeric parental building blocks. Dibenzo[b,e][1,4]diazepinone analogues might represent a potential new class of HIV PIs.

Mutations in HIV-1 gag and pol compensate for the loss of viral fitness caused by a highly mutated protease

ABSTRACT During the last few decades, the treatment of HIV-infected patients by highly active antiretroviral therapy, including protease inhibitors (PIs), has become standard. Here, we present results of analysis of a patient-derived, multiresistant HIV-1 CRF02_AG recombinant strain with a highly mutated protease (PR) coding sequence, where up to 19 coding mutations have accumulated in the PR. The results of biochemical analysis in vitro showed that the patient-derived PR is highly resistant to most of the currently used PIs and that it also exhibits very poor catalytic activity. Determination of the crystal structure revealed prominent changes in the flap elbow region and S1/S1′ active site subsites. While viral loads in the patient were found to be high, the insertion of the patient-derived PR into a HIV-1 subtype B backbone resulted in reduction of infectivity by 3 orders of magnitude. Fitness compensation was not achieved by elevated polymerase (Pol) expression, but the introduction of patient-derived gag and pol sequences in a CRF02_AG backbone rescued viral infectivity to near wild-type (wt) levels. The mutations that accumulated in the vicinity of the processing sites spanning the p2/NC, NC/p1, and p6pol/PR proteins lead to much more efficient hydrolysis of corresponding peptides by patient-derived PR in comparison to the wt enzyme. This indicates a very efficient coevolution of enzyme and substrate maintaining high viral loads in vivo under constant drug pressure.

Crystal structures of the effector-binding domain of repressor Central glycolytic gene Regulator from Bacillus subtilis reveal ligand-induced structural changes upon binding of several glycolytic intermediates.

Expression of genes in the gapA operon encoding five enzymes for triose phosphate interconversion in Bacillus subtilis is negatively regulated by the Central glycolytic genes Regulator (CggR). CggR belongs to the large SorC/DeoR family of prokaryotic transcriptional regulators, characterized by an N‐terminal DNA‐binding domain and a large C‐terminal effector‐binding domain. When no glucose is present in growth media, CggR binds to its target DNA sequence and blocks the transcription of genes in the gapA operon. In the presence of glucose, binding of the known effector molecule fructose‐1,6‐bisphosphate abolishes this interaction. We have identified dihydroxyacetone phosphate, glucose‐6‐phosphate and fructose‐6‐phosphate as additional CggR ligands that can bind to the effector‐binding site. Crystal structures of C‐CggR, the C‐terminal effector‐binding domain of CggR, both unliganded as well as in complex with the four ligands at resolutions between 1.65 and 1.80 Å reveal unique ligand‐specific structural changes in the binding site that affect the dimer interface. Binding affinities of these ligands were determined by isothermal titration calorimetry. Chemical cross‐linking shows that CggR oligomerization is mediated through its effector‐binding domain, and that binding of the different ligands differentially affects the distribution of oligomers. Electrophoretic mobility shift assays (EMSAs) confirmed a destabilizing effect of fructose‐1,6‐bisphosphate on the CggR/DNA complex, and also showed similar effects for dihydroxyacetone phosphate. Our results suggest that CggR stability and function may be modulated by various effectors in a complex fashion.

Chasing shadows: What determines DTI metrics in gray matter regions? An in vitro and in vivo study

To characterize the relationship between superparamagnetic ferritin‐bound iron and diffusion tensor scalars in vitro, and validate the results in vivo.

Thermodynamic and structural analysis of HIV protease resistance to darunavir - analysis of heavily mutated patient-derived HIV-1 proteases.

We report enzymologic, thermodynamic and structural analyses of a series of six clinically derived mutant HIV proteases (PR) resistant to darunavir. As many as 20 mutations in the resistant PRs decreased the binding affinity of darunavir by up to 13 000‐fold, mostly because of a less favorable enthalpy of binding that was only partially compensated by the entropic contribution. X‐ray structure analysis suggested that the drop in enthalpy of darunavir binding to resistant PR species was mostly the result of a decrease in the number of hydrogen bonds and a loosening of the fit between the inhibitor and the mutated enzymes. The favorable entropic contribution to darunavir binding to mutated PR variants correlated with a larger burial of the nonpolar solvent‐accessible surface area upon inhibitor binding. We show that even very dramatic changes in the PR sequence leading to the loss of hydrogen bonds with the inhibitor could be partially compensated by the entropy contribution as a result of the burial of the larger nonpolar surface area of the mutated HIV PRs.

Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase.

Betaine-homocysteine S-methyltransferase (BHMT) catalyzes the transfer of a methyl group from betaine to l-homocysteine, yielding dimethylglycine and l-methionine. In this study, we prepared a new series of BHMT inhibitors. The inhibitors were designed to mimic the hypothetical transition state of BHMT substrates and consisted of analogues with NH, N(CH(3)), or N(CH(3))(2) groups separated from the homocysteine sulfur atom by a methylene, ethylene, or a propylene spacer. Only the inhibitor with the N(CH(3)) moiety and ethylene spacer gave moderate inhibition. This result led us to prepare two inhibitors lacking a nitrogen atom in the S-linked alkyl chain: (RS,RS)-5-(3-amino-3-carboxypropylthio)-3-methylpentanoic acid and (RS)-5-(3-amino-3-carboxypropylthio)-3,3-dimethylpentanoic acid. Both of these compounds were highly potent inhibitors of BHMT. The finding that BHMT does not tolerate a true betaine mimic within these inhibitors, especially the nitrogen atom, is surprising and evokes questions about putative conformational changes of BHMT upon the binding of the substrates/products and inhibitors.

Potent inhibition of drug-resistant HIV protease variants by monoclonal antibodies.

The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.