scholarly journals Analysis and characterization of dimerization inhibition of a multi-drug-resistant Human Immunodeficiency Virus Type 1 protease using a novel size-exclusion chromatographic approach

2009 ◽  
Vol 419 (2) ◽  
pp. 497-506 ◽  
Author(s):  
David A. Davis ◽  
Irene R. Tebbs ◽  
Sarah I. Daniels ◽  
Stephen J. Stahl ◽  
Joshua D. Kaufman ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Active Site ◽  
Active Regions ◽  
Size Exclusion ◽  
Drug Resistant ◽  
Elution Time ◽  
Size Exclusion Chromatographic ◽  
Hiv 1 ◽  
Dimerization Inhibitors

Active-site inhibitors of HIV-1 PR (protease) block viral replication by preventing viral maturation. However, HIV-1 often develops resistance to active-site inhibitors through multiple mutations in PR and therefore recent efforts have focused on inhibiting PR dimerization as an alternative approach. Dimerization inhibitors have been identified using kinetic analysis, but additional characterization of the effect of these inhibitors on PR by physical methods has been difficult. In the present study, we identified a PRMDR (multi-drug-resistant HIV-1 PR) that was highly resistant to autoproteolysis. Using this PR and a novel size-exclusion chromatographic approach that incorporated fluorescence and MS detection, we were able to demonstrate inhibition of dimerization using P27 (peptide 27), a peptide dimerization inhibitor of PR previously identified on the basis of kinetic analysis. Incubation of PRMDR with P27, or other dimerization inhibitors, led to a dose- and time-dependent formation of PR monomers based on the change in elution time by size exclusion and its similar elution time to engineered forms of monomeric PR, namely PRT26A and glutathionylated PR. In contrast, incubation of PRMDR with a potent active-site inhibitor did not change the elution time for the PRMDR dimer. The monomeric PR induced by P27 had fluorescent characteristics which were consistent with unfolded PR. Structure–activity studies identified the active regions of P27 and experiments were performed to examine the effect of other dimerization inhibitors on PR. The present study is the first characterization of dimerization inhibition of PRMDR, a prime target for these inhibitors, using a novel size-exclusion chromatographic approach.

scholarly journals Characterization of a monoclonal antibody directed against the biologically active site of human interleukin 1.

1986 ◽  
Vol 163 (2) ◽  
pp. 463-468 ◽  
Author(s):  
A Köck ◽  
M Danner ◽  
B M Stadler ◽  
T A Luger
Keyword(s):  
Monoclonal Antibody ◽  
Active Site ◽  
Biological Effects ◽  
Interleukin 1 ◽  
Biologically Active ◽  
Size Exclusion ◽  
Fibroblast Proliferation ◽  
Initial Activity ◽  
Exclusion Chromatography

Human IL-1 was successfully used to produce an anti-IL-1 mAb. Anti-IL-1 (IgG2a) blocked IL-1-mediated thymocyte and fibroblast proliferation, but did not interfere with the biological effects of other lymphokines, such as IL-2 or IL-3. The antibody immunoprecipitated biosynthetically radiolabeled 33, 17, and 4 kD IL-1. An immunoadsorbent column yielded 20% of initial activity, and upon HPLC size-exclusion chromatography, affinity-purified IL-1 had a molecular mass of approximately 4 kD. These results provide first evidence of a monoclonal anti-IL-1 that reacts with different species of IL-1 and apparently binds to an epitope close to the active site of IL-1. Thus, anti-IL-1 IgG may be very helpful for further investigations of the molecular as well as biological characteristics of IL-1 and related mediators.

scholarly journals Lipopeptides as Dimerization Inhibitors of HIV-1 Protease

1999 ◽  
Vol 380 (5) ◽  
pp. 593-596 ◽  
Author(s):  
H. J. Schramm ◽  
E. De Rosny ◽  
M. Reboud-Ravaux ◽  
J. Büttner ◽  
A. Dick ◽  
...  
Keyword(s):  
Active Site ◽  
Structural Features ◽  
Hiv Protease ◽  
Blocking Group ◽  
Aids Therapy ◽  
Inhibitory Power ◽  
Natural Amino Acids ◽  
Enzyme Test ◽  
Hiv 1 ◽  
Dimerization Inhibitors

Abstract In AIDS therapy, attempts have been made to inhibit the virus-encoded enzymes, e.g. HIV-1 protease, using active site-directed inhibitors. This approach is questionable, however, due to virus mutations and the high toxicity of the drugs. An alternative method to inhibit the dimeric HIV protease is the targeting of the interface region of the protease subunits in order to prevent subunit dimerization and enzyme activity. This approach should be less prone to inactivation by mutation. A list of improved ‘dimerization inhibitors’ of HIV-1 protease is presented. The main structural features are a short ‘interface’ peptide segment, including non-natural amino acids, and an aliphatic N-terminal blocking group. The high inhibitory power of some of the lipopeptides [e.g. palmitoyl-Tyr-Glu-Leu-OH, palmitoyl-Tyr-Glu-(L-thyronine)-OH, palmitoyl-Tyr-Glu-(L-biphenyl-alanine)-OH] with low nanomolar Ki valuesin the enzyme test suggests that mimetics with good bio-availability can be derived for AIDS therapy.

scholarly journals Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Paul L. Boyer ◽  
Kevin Melody ◽  
Steven J. Smith ◽  
Linda L. Dunn ◽  
Chris Kline ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Active Site ◽  
Nucleoside Analogs ◽  
Transcriptase Inhibitor ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Hydrophobic Region ◽  
Nnrti Resistance ◽  
Hiv 1

ABSTRACTTwo mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the “primer grip” region and could be influenced by the M230I mutation.IMPORTANCEAlthough antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

scholarly journals Anti-HIV Activity of MDL 74968, a Novel Acyclonucleotide Derivative of Guanine: Drug Resistance and Drug Combination Effects in Vitro

1996 ◽  
Vol 7 (5) ◽  
pp. 253-260 ◽  
Author(s):  
D.L. Taylor ◽  
S.P. Ahmed ◽  
T.M. Brennan ◽  
J.-F. Navé ◽  
P. Casara ◽  
...  
Keyword(s):  
Nucleoside Analogues ◽  
Drug Resistant ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
Experimental Protocols ◽  
Hiv 1

MDL 74968 (9-[2-methylidene-3-(phosphonomethoxy)-propyl]guanine), a novel acyclonucleotide derivative of guanine, inhibited human immunodeficiency virus type 1 (HIV-1) replication in vitro with activity comparable to that of adefovir (PMEA; 9-(2-phosphonomethoxyethyl)adenine). MDL 74968 was investigated in combination with two licensed nucleoside analogues, zidovudine and didanosine, using a cell viability assay, and drug interactions were evaluated by the isobologram technique, by calculating combination indices and by the MacSynergy™ program. Inhibition of HIV-1 replication was only additive in both cases. MDL 74968 had equivalent antiviral activity against strains of HIV-1 HXB2 engineered to have mutations which conferred resistance to the nucleoside analogues lamivudine, didanosine and zidovudine and the non-nucleoside inhibitor of reverse transcriptase (RT) nevirapine, as against the wild type strain. Continued passage of HIV-1 RF in C8166 cells in the presence of MDL 74968 for 5 months (30 passages) failed to select drug resistant mutants. Continued passage of virus in the presence of the same concentration of adefovir for the same length of time selected a virus in a single culture, which was 3-fold resistant to adefovir and cross-resistant to MDL 74968. Genotypic characterization of this virus revealed a lysine to arginine exchange (AAA to AGA) at position 65 in the RT gene. This virus was not cross-resistant to either zidovudine or nevirapine but showed reduced sensitivity to zalcitabine, didanosine and lamivudine. Continued passage of HIV-1 RF in the presence of nevirapine or zidovudine, using similar experimental protocols selected drug resistant viruses after eight and 17 passages, respectively, but these viruses remained sensitive to adefovir and MDL 74968.

Size exclusion chromatographic characterization of sodium hyaluronate fractions prepared by high energetic sonication

1993 ◽  
Vol 37 (1-2) ◽  
pp. 20-22 ◽  
Author(s):  
E. Orviský ◽  
L. Šoltés ◽  
P. Chabreček ◽  
I. Novák ◽  
M. Stančíková
Keyword(s):  
Sodium Hyaluronate ◽  
Size Exclusion ◽  
Size Exclusion Chromatographic ◽  
Chromatographic Characterization

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease

1999 ◽  
Vol 263 (1) ◽  
pp. 238-244 ◽  
Author(s):  
Bhuvaneshwari Mahalingam ◽  
John M. Louis ◽  
Charles C. Reed ◽  
Jill M. Adomat ◽  
Jennifer Krouse ◽  
...  
Keyword(s):  
Kinetic Analysis ◽  
Drug Resistant ◽  
Resistant Mutants ◽  
Hiv 1

scholarly journals Defective Hydrophobic Sliding Mechanism and Active Site Expansion in HIV-1 Protease Drug Resistant Variant Gly48Thr/Leu89Met: Mechanisms for the Loss of Saquinavir Binding Potency

Biochemistry ◽  
2015 ◽  
Vol 54 (2) ◽  
pp. 422-433 ◽  
Author(s):  
Nathan E. Goldfarb ◽  
Meray Ohanessian ◽  
Shyamasri Biswas ◽  
T. Dwight McGee ◽  
Brian P. Mahon ◽  
...  
Keyword(s):  
Active Site ◽  
Drug Resistant ◽  
Resistant Variant ◽  
Sliding Mechanism ◽  
Hiv 1 ◽  
Drug Resistant Variant
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