Biomedical subjects

B Selmi

Publications and source records attributed to B Selmi.

6 recordsLinked to original sources

PubMed2004

Structural determinants and molecular mechanisms for the resistance of HIV-1 RT to nucleoside analogues.

The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is an RNA- and DNA-dependent DNA polymerase capable of copying the viral genome before it gets integrated into the human host DNA. Hence, HIV-1 RT plays a major role in viral replication and represents a key target for anti-AIDS treatments. Amongst the eleven licensed drugs that inhibit RT, eight are chain-terminating nucleoside analogues (NRTIs) that compete with their natural counterparts during the DNA polymerization process. Unfortunately, under therapeutic pressure, the HIV-1 inevitably develops resistance to these inhibitors by accumulating mutations in the viral pol gene encoding RT. Mechanisms for this resistance can be sorted in two categories, depending on the nature of the drug and the selected mutations. The first category includes mutations involving a specific alteration of the discrimination between natural nucleotides and NRTIs. The second category includes mutations able to promote the removal of the incorporated NRTI and thus repair the nascent DNA chain. This review summarizes the modes of inhibition of HIV-1 RT with NRTIs, and describes the mechanisms of resistance to these drugs, based on enzymatic data correlated to crystal structures and molecular models involving HIV-1 RT. We also give insights into different aspects of resistance such as antagonistic mutations, replication capacity, and the implications for a rational, structure-based drug design.

Animals↗

PubMed2001

Mechanism-based suppression of dideoxynucleotide resistance by K65R human immunodeficiency virus reverse transcriptase using an alpha-boranophosphate nucleoside analogue.

The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2',3'-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2',3'-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo. A decreased catalytic rate constant k(pol) mostly accounts for the lower incorporation efficiency observed for 2',3'-dideoxynucleotides. Examination of the crystal structure of the RT.DNA.dNTP complex suggested that both the charge at position 65 and the 3'-OH of the incoming nucleotide act in synergy during the creation of the phosphodiester bond, resulting in a more pronounced decreased catalytic rate constant for 2',3'-dideoxynucleotides than for dNTPs. This type of intramolecular activation of the leaving phosphate by the 3'-OH group appears to be conserved in several nucleotide phosphotransferases. These data were used to design dideoxynucleotide analogues targeting K65R RT specifically. alpha-Boranophosphate ddATP was found to be a 2-fold better substrate than dATP and inhibited DNA synthesis by K65R RT 153-fold better than ddATP. This complete suppression of drug resistance at the nucleotide level could serve for other reverse transcriptases for which drug resistance is achieved at the catalytic step.

Amino Acid Substitution↗

PubMed2001

An integrated system to study multiply substituted human immunodeficiency virus type 1 reverse transcriptase.

We describe a gene system allowing the facile production of multiply substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophagetropic, non-laboratory-adapted human immunodeficiency virus type 1 (HIV-1) AD8 strain. Thirteen unique silent restriction sites were introduced in the pol gene encoding HIV-1 RT, allowing easy introduction of mutations. To simplify genetic manipulation and generate p66/p51 heterodimers in Escherichia coli, a gene construct of the viral protease alone was optimized for expression from a separate vector carrying a p15A origin of replication. Active-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzyme than that obtained by alternate methods. To facilitate phenotype/genotype correlations, the modified RT gene was designed to be easily reintroduced into a recombinant proviral AD8 HIV-1 DNA. Infectious viruses made from this vector were undistinguishable from wild-type AD8 HIV-1, an isolate able to infect peripheral blood mononuclear cells and macrophages. Thus, the pol gene can tolerate many silent mutations in the polymerase domain without affecting the functionality of the HIV-1 genome. The system was validated biochemically and virologically using the V75T substitution associated with stavudine resistance.

Amino Acid Substitution↗

PubMed2000

The valine-to-threonine 75 substitution in human immunodeficiency virus type 1 reverse transcriptase and its relation with stavudine resistance.

The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5'-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site.

Adenosine Triphosphate↗

PubMed2000

Phosphorylation of AZT-resistant human immunodeficiency virus type 1 reverse transcriptase by casein kinase II in vitro: effects on inhibitor sensitivity.

Casein kinase II (CKII) phosphorylates wild-type (WT) recombinant reverse transcriptase (RT) mainly in the p66 subunit in vitro. Phosphorylation of T215F RT and D67N/K70R/T215F/K219Q RT (AZT-resistant RT) in vitro increases discrimination against AZTTP 2. 5- and 3.6-fold, respectively. This in vitro resistance can be reversed by treatment of phosphorylated AZT-resistant RT with phosphatase. Phosphorylation has no effect on WT RT. Terminal transferase activity of RT is selectively suppressed on phosphorylated AZT-resistant RT. Resistance to phosphonoformic acid (PFA, foscarnet) increases 3-fold upon phosphorylation of AZT-resistant RT. Although T215, the most important residue for AZT-resistance, is part of a CKII consensus target site, serines are primarily phosphorylated relative to threonines. Mutational analysis shows that phosphorylation can be reduced to 10% that of WT when amino-acid changes are introduced both in the "fingers" subdomain and motif D. These results suggest that phosphorylation of RT might be one factor involved in drug resistance in vivo.

Amino Acid Motifs↗

PubMed2000

Amyloglucosidase hydrolysis of high-pressure and thermally gelatinized corn and wheat starches.

The study of glucose production using amyloglucosidase as a biocatalyst was carried out using high-pressure and thermally gelatinized corn and wheat starches. For corn starch, the measured initial rate of glucose production obtained from thermal gelatinization is faster than that obtained from the two high-pressure treatments, but the equilibrium yield of glucose was found to be similar for the three treatments. High-pressure treatments of wheat starch significantly improve the equilibrium yield of glucose compared with those obtained from the thermally gelatinized wheat starch. This difference has been related to the formation of amylose-lipid complexes during heating and could also explain previous physicochemical differences observed between high-pressure and thermally gelatinized starch.

Catalysis↗