ABSTRACT
We have recently demonstrated that metalloporphyrins are potent inhibitors of both human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2) reverse transcriptases (RTs) [Argyris, E.G., Vanderkooi, J.M., Venkateswaran, P.S., Kay, B.K., and Paterson, Y. (1999) J. Biol. Chem. 274, 1549-1556]. In addition, by screening a phage peptide library we discovered that a peptide with sequence similarity to residues 398-407 from the connection subdomain of HIV RTs binds heme. These findings suggested that this highly conserved region may be the binding site for metalloporphyrins and a novel site for inhibition of enzymatic activity. Our most recent data presented here confirm this suggestion. Screening of HIV-1 RT 398-407 peptide analogs by fluorescence assays demonstrates that Trp residues at positions 401 and 402 are important for heme binding. Furthermore, site-directed mutagenesis of these residues verified these findings and indicated that heme inhibits HIV-1 RT by binding on the connection subdomain of the p66 subunit of the enzyme but not on the p51 subunit. This was also confirmed by analyzing the binding affinities of heme for mutant HIV-1 RT heterodimers, using intrinsic fluorescence assays. The clear identification of the connection domain as a novel inhibition site is crucial in understanding the mechanism of heme binding and enzymatic inhibition and will facilitate the generation of novel porphyrin-based inhibitors of RT.
Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/genetics , Heme/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , Heme/metabolism , Metalloporphyrins/metabolism , Metalloporphyrins/pharmacology , Microscopy, Fluorescence , Models, Molecular , Mutagenesis, Site-Directed , Oligopeptides/metabolism , Protein Structure, Tertiary , Reverse Transcriptase Inhibitors/metabolism , Tryptophan/metabolismABSTRACT
We have shown that heme and zinc protoporphyrin inhibit both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs) and, in combination with other nucleoside and non-nucleoside inhibitors, exert an additive effect on HIV-1 RT inhibition. Screening of a phage peptide library against heme resulted in the isolation of a peptide with sequence similarity to sequence 398-407 from the connection subdomain of both HIV-1 and HIV-2 RTs, suggesting that this highly conserved region of HIV RTs corresponds to the binding site for metalloporphyrins and a new site for inhibition of enzyme activity. Inclusion of a synthetic peptide corresponding to the exact sequence 398-407 of HIV-1 RT in RT inhibition assays had a protective effect on metalloporphyrin inhibition, as it was able to reverse the inhibitory effect of both metalloporphyrins on HIV-1 RT activity. Furthermore, intrinsic fluorescence assays indicated that these metalloporphyrins bind to synthetic peptide 398-407 as well as to intact dimeric HIV-1 RT. The identification of this novel inhibition site will help to expand our understanding of the mode of action of metalloporphyrins in RT inhibition and will assist in the design and development of more potent metalloporphyrin RT inhibitors for the management of HIV infection.
