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Human immunodeficiency virus type 1 proteinase resistance to symmetric cyclic urea inhibitor analogs
U Nillroth1, L Vrang, P O Markgren
1Department of Biochemistry, Uppsala University, Sweden.
Abstract:
Resistant virus was isolated from virus propagated in cell culture in the presence of the human immunodeficiency virus type 1 (HIV-1) proteinase inhibitor DMP 323, Ro 31-8959, or A-75925. The proteinase gene of resistant virus was sequenced, and key mutations (G48V, V82A, I84V, L90M, and G48V/L90M) were introduced into clones used for the expression, purification, and further characterization of the enzyme. The mutant enzymes were all less active than the wild-type enzyme, as judged by k(cat) and k(cat)/Km values. L90M had a lower Km than the wild type, whereas the G48V/L90M double mutant had an increased Km compared with that of the wild type, contributing to a 10-fold reduction in the k(cat)/Km. Vitality values were used to show that the enzyme of the I84V mutant is the enzyme most resistant to the two cyclic urea inhibitors DMP 323 and AHA 008. Virus with the same mutation is also resistant, although the double mutation L10F/I84V confers even greater resistance. All of these mutants are more resistant to DMP 323 than to AHA 008. The resistance of the I84V mutant may be attributed to a loss of van der Waals interactions with the inhibitor, since the larger amino acid side chain involved in the interaction is replaced by a smaller side chain. This is supported by the lower level of resistance to AHA 008 that was observed. The phenyl groups of AHA 008 should protrude deeper into the S1 and S1' subsites than those of the smaller compound DMP 323, reducing the loss of interaction energy. These results reveal that small structural modifications of inhibitors that do not affect the inhibitory effect on wild-type virus can influence the inhibition of resistant strains. This is of importance for optimizing drugs with respect to their potency and resistance.
Insights
Mutations in human immunodeficiency virus type 1 (HIV-1) proteinase confer resistance to inhibitors like DMP 323. Understanding these mutations, such as I84V, is key to developing more effective antiviral drugs against resistant HIV-1 strains.
Area of Science:
- Virology
- Drug Resistance
- Biochemistry
Background:
- Human immunodeficiency virus type 1 (HIV-1) proteinase inhibitors are crucial for antiretroviral therapy.
- Emergence of drug-resistant HIV-1 strains necessitates continuous drug development and optimization.
- Specific mutations in the HIV-1 proteinase gene can lead to reduced susceptibility to existing inhibitors.
Purpose of the Study:
- To investigate the molecular basis of resistance to HIV-1 proteinase inhibitors.
- To characterize the activity and resistance profiles of HIV-1 proteinase mutants.
- To inform the design of next-generation HIV-1 inhibitors with improved resistance profiles.
Main Methods:
- Isolation of drug-resistant HIV-1 strains in cell culture.
- Sequencing of the HIV-1 proteinase gene to identify resistance mutations.
- Site-directed mutagenesis to introduce specific mutations into the proteinase gene.
- Enzyme kinetics (k(cat), k(cat)/Km, Km) to assess mutant enzyme activity.
- Inhibition assays using cyclic urea inhibitors (DMP 323, AHA 008) to determine resistance levels.
Main Results:
- Key mutations (G48V, V82A, I84V, L90M, G48V/L90M) were identified in resistant HIV-1 strains.
- Mutant proteinase enzymes exhibited reduced catalytic activity compared to wild-type.
- The I84V mutant showed the highest resistance to DMP 323 and AHA 008.
- Resistance was generally higher to DMP 323 than to AHA 008.
- Structural analysis suggests that the I84V mutation reduces van der Waals interactions with inhibitors.
Conclusions:
- Specific mutations in HIV-1 proteinase significantly impact inhibitor susceptibility.
- The I84V mutation confers substantial resistance, potentially due to altered binding interactions.
- Even minor structural changes in inhibitors can influence efficacy against resistant HIV-1 strains.
- These findings are critical for optimizing existing drugs and designing novel inhibitors to overcome drug resistance.