FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2022901458> ?p ?o ?g. }

• W2022901458 endingPage "4920" @default.
• W2022901458 startingPage "4914" @default.
• W2022901458 abstract "Enfuvirtide (T-20) is a fusion inhibitor that suppresses replication of human immunodeficiency virus (HIV) variants with multi-drug resistance to reverse transcriptase and protease inhibitors. It is a peptide derived from the C-terminal heptad repeat (C-HR) of HIV-1 gp41, and it prevents interactions between the C-HR and the N-terminal HR (N-HR) of gp41, thus interfering with conformational changes that are required for viral fusion. However, prolonged therapies with T-20 result in the emergence of T-20-resistant strains that contain primary mutations such as N43D in the N-HR of gp41 (where T-20 and C-HR bind) that help the virus escape at a fitness cost. Such variants often go on to acquire a secondary mutation, S138A, in the C-HR of gp41 region that corresponds to the sequence of T-20. We demonstrate here that the role of S138A is to compensate for the impaired fusion kinetics of HIV-1s carrying primary mutations that abrogate binding of T-20. To preempt this escape strategy, we designed a modified T-20 variant containing the S138A substitution and showed that it is a potent inhibitor of both T-20-sensitive and T-20-resistant viruses. Circular dichroism analysis revealed that the S138A provided increased stability of the 6-helix bundle. We validated our approach on another fusion inhibitor, C34. In this case, we designed a variant of C34 with the secondary escape mutation N126K and showed that it can effectively inhibit replication of C34-resistant HIV-1. These results prove that it is possible to design improved peptide-based fusion inhibitors that are efficient against a major mechanism of drug resistance. Enfuvirtide (T-20) is a fusion inhibitor that suppresses replication of human immunodeficiency virus (HIV) variants with multi-drug resistance to reverse transcriptase and protease inhibitors. It is a peptide derived from the C-terminal heptad repeat (C-HR) of HIV-1 gp41, and it prevents interactions between the C-HR and the N-terminal HR (N-HR) of gp41, thus interfering with conformational changes that are required for viral fusion. However, prolonged therapies with T-20 result in the emergence of T-20-resistant strains that contain primary mutations such as N43D in the N-HR of gp41 (where T-20 and C-HR bind) that help the virus escape at a fitness cost. Such variants often go on to acquire a secondary mutation, S138A, in the C-HR of gp41 region that corresponds to the sequence of T-20. We demonstrate here that the role of S138A is to compensate for the impaired fusion kinetics of HIV-1s carrying primary mutations that abrogate binding of T-20. To preempt this escape strategy, we designed a modified T-20 variant containing the S138A substitution and showed that it is a potent inhibitor of both T-20-sensitive and T-20-resistant viruses. Circular dichroism analysis revealed that the S138A provided increased stability of the 6-helix bundle. We validated our approach on another fusion inhibitor, C34. In this case, we designed a variant of C34 with the secondary escape mutation N126K and showed that it can effectively inhibit replication of C34-resistant HIV-1. These results prove that it is possible to design improved peptide-based fusion inhibitors that are efficient against a major mechanism of drug resistance. HIV-1 2The abbreviations used are: HIV, human immunodeficiency virus; T-20, enfuvirtide; HR, heptad repeat; MAGI, multinuclear activation of galactosidase indicator; EC50, 50% effective concentration; Tm, melting temperature; CD, circular dichroism; shRNA, short hairpin RNA; WT, wild-type.2The abbreviations used are: HIV, human immunodeficiency virus; T-20, enfuvirtide; HR, heptad repeat; MAGI, multinuclear activation of galactosidase indicator; EC50, 50% effective concentration; Tm, melting temperature; CD, circular dichroism; shRNA, short hairpin RNA; WT, wild-type. entry into the target cells is mediated by two envelope glycoproteins, gp120 and gp41, that form a trimeric gp120·gp41 complex. After binding of gp120 to the CD4 receptor and CCR5 (or CXCR4) coreceptor on the surface of the target cell, the gp41 trimer forms an extended conformation of the three helices that allows a hydrophobic fusion peptide to be inserted into the target cell membrane, generating an intermediate that is anchored to both cellular and viral membranes. After this step, the gp41 is believed to start refolding to a more stable 6-helix bundle composed of the α-helical trimer of the N-terminal heptad repeat (N-HR) folded into an anti-parallel conformation with the three C-terminal heptad repeats (C-HR) (1Chan D.C. Kim P.S. Cell. 1998; 93: 681-684Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 2Weiss C.D. AIDS Rev. 2003; 5: 214-221PubMed Google Scholar). This refolding brings the viral and cellular membranes together to catalyze fusion. The transition of the extended intermediate to the 6-helix bundle can be inhibited by the addition of exogenous peptides derived from gp41 C-HR (Fig. 1A) that prevent the formation of the 6-helix bundle and inhibit the HIV-1 fusion with the target cells (3Jiang S. Lin K. Strick N. Neurath A.R. Nature. 1993; 365: 113Crossref PubMed Scopus (475) Google Scholar, 4Chan D.C. Chutkowski C.T. Kim P.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15613-15617Crossref PubMed Scopus (484) Google Scholar, 5Dwyer J.J. Wilson K.L. Davison D.K. Freel S.A. Seedorff J.E. Wring S.A. Tvermoes N.A. Matthews T.J. Greenberg M.L. Delmedico M.K. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12772-12777Crossref PubMed Scopus (219) Google Scholar, 6Otaka A. Nakamura M. Nameki D. Kodama E. Uchiyama S. Nakamura S. Nakano H. Tamamura H. Kobayashi Y. Matsuoka M. Fujii N. Angew. Chem. Int. Ed. Engl. 2002; 41: 2937-2940Crossref PubMed Google Scholar). T-20, a 36-amino acid peptide derived from C-HR, effectively suppresses in vivo replication of HIV-1 resistant to inhibitors of reverse transcriptase and protease (7Lalezari J.P. Henry K. O'Hearn M. Montaner J.S. Piliero P.J. Trottier B. Walmsley S. Cohen C. Kuritzkes D.R. Eron Jr., J.J. Chung J. DeMasi R. Donatacci L. Drobnes C. Delehanty J. Salgo M. N. Engl. J. Med. 2003; 348: 2175-2185Crossref PubMed Scopus (825) Google Scholar, 8Lazzarin A. Clotet B. Cooper D. Reynes J. Arasteh K. Nelson M. Katlama C. Stellbrink H.J. Delfraissy J.F. Lange J. Huson L. DeMasi R. Wat C. Delehanty J. Drobnes C. Salgo M. N. Engl. J. Med. 2003; 348: 2186-2195Crossref PubMed Scopus (608) Google Scholar). However, HIV-1 variants resistant to T-20 have recently emerged carrying primary mutations in the Leu-33–Leu-45 region of the N-HR domain (9Baldwin C.E. Berkhout B. Retrovirology. 2006; 3: 84Crossref PubMed Scopus (17) Google Scholar, 10Cabrera C. Marfil S. Garcia E. Martinez-Picado J. Bonjoch A. Bofill M. Moreno S. Ribera E. Domingo P. Clotet B. Ruiz L. AIDS. 2006; 20: 2075-2080Crossref PubMed Scopus (40) Google Scholar, 11Labrosse B. Morand-Joubert L. Goubard A. Rochas S. Labernardiere J.L. Pacanowski J. Meynard J.L. Hance A.J. Clavel F. Mammano F. J. Virol. 2006; 80: 8807-8819Crossref PubMed Scopus (48) Google Scholar, 12Mink M. Mosier S.M. Janumpalli S. Davison D. Jin L. Melby T. Sista P. Erickson J. Lambert D. Stanfield-Oakley S.A. Salgo M. Cammack N. Matthews T. Greenberg M.L. J. Virol. 2005; 79: 12447-12454Crossref PubMed Scopus (111) Google Scholar, 13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar, 14Perez-Alvarez L. Carmona R. Ocampo A. Asorey A. Miralles C. Perez de Castro S. Pinilla M. Contreras G. Taboada J.A. Najera R. J. Med. Virol. 2006; 78: 141-147Crossref PubMed Scopus (41) Google Scholar, 15Xu L. Pozniak A. Wildfire A. Stanfield-Oakley S.A. Mosier S.M. Ratcliffe D. Workman J. Joall A. Myers R. Smit E. Cane P.A. Greenberg M.L. Pillay D. Antimicrob. Agents Chemother. 2005; 49: 1113-1119Crossref PubMed Scopus (155) Google Scholar). Among them, V38A and N43D seem to be major primary mutations for T-20 resistance. Meanwhile, a secondary mutation at the C-HR region (S138A) has been reported to enhance T-20 resistance with an as yet undefined mechanism (9Baldwin C.E. Berkhout B. Retrovirology. 2006; 3: 84Crossref PubMed Scopus (17) Google Scholar, 14Perez-Alvarez L. Carmona R. Ocampo A. Asorey A. Miralles C. Perez de Castro S. Pinilla M. Contreras G. Taboada J.A. Najera R. J. Med. Virol. 2006; 78: 141-147Crossref PubMed Scopus (41) Google Scholar, 15Xu L. Pozniak A. Wildfire A. Stanfield-Oakley S.A. Mosier S.M. Ratcliffe D. Workman J. Joall A. Myers R. Smit E. Cane P.A. Greenberg M.L. Pillay D. Antimicrob. Agents Chemother. 2005; 49: 1113-1119Crossref PubMed Scopus (155) Google Scholar) (Fig. 1B). The mechanism of resistance to C34, another C-HR peptide-based inhibitor of HIV fusion, has been the subject of multiple studies (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar, 16Armand-Ugon M. Gutierrez A. Clotet B. Este J.A. Antiviral Res. 2003; 59: 137-142Crossref PubMed Scopus (66) Google Scholar). Because of a 22-amino acid overlap between the T-20 and C34 peptides (Fig. 1B), HIV-1 has developed primary mutations for C34 resistance in vitro at the identical Leu-33–Leu-45 region of the peptides. During in vitro selection of C34 resistance, we identified a mutation in the C-HR domain, N126K, that is also observed in some T-20-resistant clinical variants (10Cabrera C. Marfil S. Garcia E. Martinez-Picado J. Bonjoch A. Bofill M. Moreno S. Ribera E. Domingo P. Clotet B. Ruiz L. AIDS. 2006; 20: 2075-2080Crossref PubMed Scopus (40) Google Scholar, 15Xu L. Pozniak A. Wildfire A. Stanfield-Oakley S.A. Mosier S.M. Ratcliffe D. Workman J. Joall A. Myers R. Smit E. Cane P.A. Greenberg M.L. Pillay D. Antimicrob. Agents Chemother. 2005; 49: 1113-1119Crossref PubMed Scopus (155) Google Scholar, 17Baldwin C.E. Sanders R.W. Deng Y. Jurriaans S. Lange J.M. Lu M. Berkhout B. J. Virol. 2004; 78: 12428-12437Crossref PubMed Scopus (129) Google Scholar). We showed that N126K conferred resistance to C34 by compensating for the impaired intra-gp41 interaction by a primary mutation, I37K (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar). N126K was initially identified in background of V38A, another primary mutation, for T-20 resistance in vivo (17Baldwin C.E. Sanders R.W. Deng Y. Jurriaans S. Lange J.M. Lu M. Berkhout B. J. Virol. 2004; 78: 12428-12437Crossref PubMed Scopus (129) Google Scholar). Baldwin et al. (17Baldwin C.E. Sanders R.W. Deng Y. Jurriaans S. Lange J.M. Lu M. Berkhout B. J. Virol. 2004; 78: 12428-12437Crossref PubMed Scopus (129) Google Scholar, 18Baldwin C. Berkhout B. J. Virol. 2008; 82: 7735-7740Crossref PubMed Scopus (23) Google Scholar) demonstrated a striking T-20-dependent replication phenotype in the V38A/N126K variant and proposed that T-20 acts as a safety pin to prevent premature formation of helical bundle, as N126K enhanced binding capacity of the introduced C-HR to N36 with V38A. Taken together, these studies suggest that mutations in the C-HR serve as secondary mutations. In this study we show that the main role of secondary mutations that follow the appearance of primary mutations during treatment with peptide-based fusion inhibitors is to compensate for the impairment in replication kinetics that is caused by the primary mutations (supplemental Fig. 1). Based on this finding we hypothesized that analogs of T-20 carrying substitutions corresponding to secondary T-20 resistance mutations should be active against both wild-type and T-20-resistant viruses containing primary mutations. Indeed, our results confirmed our hypothesis and showed that T-20 with the S138A substitution (T-20S138A) has a strong anti-HIV-1 activity even against T-20-resistant clones. Moreover, we demonstrate that this restoration is concomitant to improved binding of C-HRS138A to N-HRN43D, suggesting that our approach utilizing the resistance-associated mutations to design peptides may provide useful broad insights into effective peptide-based therapies. Cells and Viruses—MT-2 cells were grown in RPMI 1640 medium. 293T cells were grown in Dulbecco's modified Eagle's medium-based culture medium. HeLa-CD4-LTR-β-gal cells were kindly provided by M. Emerman through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health (Bethesda, MD) and were used for the drug susceptibility assay as described previously (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar, 19Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Crossref PubMed Google Scholar, 20Maeda Y. Venzon D.J. Mitsuya H. J. Infect. Dis. 1998; 177: 1207-1213Crossref PubMed Scopus (119) Google Scholar). An HIV-1 infectious clone, pNL4–3 (21Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar), was used for generation of HIV-1 variants. Antiviral Agents—The peptides used in this study were synthesized as described previously (6Otaka A. Nakamura M. Nameki D. Kodama E. Uchiyama S. Nakamura S. Nakano H. Tamamura H. Kobayashi Y. Matsuoka M. Fujii N. Angew. Chem. Int. Ed. Engl. 2002; 41: 2937-2940Crossref PubMed Google Scholar). Determination of Drug Susceptibility of HIV-1—The peptide sensitivity of infectious clones was determined by the multinuclear activation of galactosidase indicator (MAGI) assay as described previously (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar). Briefly, the target cells (HeLa-CD4-LTR-β-gal; 104 cells/well) were plated in 96-well flat microtiter culture plates. On the following day the cells were inoculated with the HIV-1 clones (60 MAGI unit/well, giving 60 blue cells after 48 h of incubation) and cultured in the presence of various concentrations of drugs in fresh medium. Forty-eight hours after viral exposure, all the blue cells stained with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) were counted in each well. The activity of test compounds was determined as the concentration that blocked HIV-1 replication by 50% (50% effective concentration, (EC50). Generation of Recombinant HIV-1 Clones—Recombinant infectious HIV-1 clones, carrying various mutations, were generated as described previously (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar). Each molecular clone was transfected into 293T cells with TransIT® (Madison, WI). After 48 h, the supernatants were harvested and stored at –80 °C until use. Circular Dichroism Spectroscopy—Each peptide (10 μm) was mixed with 10 mm phosphate-buffered saline, pH 7.4, and the data were collected using a Jasco spectrometer (Model J-710; Jasco, Tokyo, Japan) equipped with a thermoelectric temperature controller. The thermal stability was assessed by monitoring the change in the circular dichroism signal at 222 nm. The midpoint of the thermal unfolding transition (melting temperature, (Tm) of each complex was determined as described previously (6Otaka A. Nakamura M. Nameki D. Kodama E. Uchiyama S. Nakamura S. Nakano H. Tamamura H. Kobayashi Y. Matsuoka M. Fujii N. Angew. Chem. Int. Ed. Engl. 2002; 41: 2937-2940Crossref PubMed Google Scholar). Viral Replication Kinetics Assay—MT-2 cells (105 cells/3 ml) were infected with each virus preparation (1000 MAGI unit) for 16 h. The infected cells were then washed and cultured in a final volume of 3 ml. The culture supernatants were harvested after infection on days 2–7, and the levels of p24 antigen were determined (22Hachiya A. Kodama E.N. Sarafianos S.G. Schuckmann M.M. Sakagami Y. Matsuoka M. Takiguchi M. Gatanaga H. Oka S. J. Virol. 2008; 82: 3261-3270Crossref PubMed Scopus (86) Google Scholar). For each competitive HIV-1 replication assay, two infectious clones of interest that had been previously titrated were mixed and added to MT-2 cells (105 cells/3 ml) as described previously (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar, 22Hachiya A. Kodama E.N. Sarafianos S.G. Schuckmann M.M. Sakagami Y. Matsuoka M. Takiguchi M. Gatanaga H. Oka S. J. Virol. 2008; 82: 3261-3270Crossref PubMed Scopus (86) Google Scholar) with minor modifications. To ensure that the two infectious clones being compared were of approximately equal infectivity, a fixed amount (500 MAGI unit) of one infectious clone was mixed with three different amounts (250, 500, and 1000 MAGI unit) of the other infectious clone. On day 1, one-third of the infected MT-2 cells were harvested and washed twice with phosphate-buffered saline, and the cellular DNA was extracted. The purified DNA was subjected to nested PCR and then direct DNA sequencing. The HIV-1 co-culture, which best approximated a 50:50 mixture on day 1, was further propagated. Every 3–4 days, the co-culture supernatant (100 μl) was transmitted to new uninfected MT-2 cells (5 × 105 cells/3 ml). The cells harvested at the end of each passage were subjected to direct sequencing, and the viral population change was determined. Structure Modeling of gp41 S138A Mutant Core—The gp41 core model was built using the coordinates of crystal structure of the N36/C34 complex (23Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1833) Google Scholar) (PDB code 1AIK). The coordinates of the water molecules were removed. Additionally, the hydrogen atoms were placed in optimal positions and refined by the energy minimization with the AMBER9 program (24Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6422) Google Scholar) using the FF99 force field. Ser-138 in the gp41 core model was replaced with alanine (replacement of -OH with -H), and the positions of the hydrogen atoms were refined as described above. The S138A mutant core model (N36/C34S138A complex) was further optimized by the energy minimization using the FF99 force field with the restraints on each of the three residues of N and C termini and the backbone atoms. The restraint weight was 5.0 kcal/mol Å2. Effect of Amino Acid Substitutions at 138 on Antiviral Activities—We chemically synthesized peptide analogs of T-20 with all natural amino acid substitutions at the 138 position (T-20S138X) and evaluated them for their ability to inhibit three major T-20-resistant clones using the MAGI assay (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar) (Table 1). The results indicated that only T-20S138A inhibited replication of T-20-resistant clones as efficiently as the wild-type clone. Substitution to glycine enhanced T-20 activity, but unlike T-20S138A, T-20S138G reduced its activity against T-20-resistant clones by ∼2–3-fold as compared with the parental peptide, T-20. Substitutions to hydrophobic amino acids leucine, isoleucine, and methionine maintained their anti-HIV-1 activity; however, those to valine reduced anti-HIV-1 activity to T-20-resistant clones. The proline substitution drastically decreased the anti-HIV-1 activity of the peptide inhibitors.TABLE 1Antiviral activity of T-20-derived peptides against T-20-resistant gp41 recombinant virusesEC50HIV-1WTaTo improve the replication kinetics, D36G mutation, observed in the majority of HIV-1 strains, was introduced into the NL4-3 background used in this study (reference virus).HIV-1V38AHIV-1N43DHIV-1N43D/S138AnmT-20>2.4 ± 0.623 ± 8.2 (9.6)49 ± 10 (20)84 ± 16 (35)Small T-20S138G1.3 ± 0.5 (0.5)65 ± 8.8 (27)141 ± 26 (59)185 ± 68 (77) T-20S138A0.6 ± 0.1 (0.3)3.6 ± 1.7 (1.5)3.5 ± 0.9 (1.5)3.2 ± 1.0 (1.3)Hydrophobic T-20S138V0.4 ± 0.2 (0.2)31 ± 14 (13)22 ± 3.5 (9.2)23 ± 5.7 (9.6) T-20S138L0.7 ± 0.1 (0.3)13 ± 6 (5.4)2.9 ± 0.7 (1.2)2.2 ± 0.4 (0.9) T-20S138I0.5 ± 0.1 (0.2)4.9 ± 2 (2)2.9 ± 0.8 (1.2)2.4 ± 0.6 (1) T-20S138M0.7 ± 0.2 (0.3)4.4 ± 0.1 (1.8)1.7 ± 0.5 (0.7)1.2 ± 0.4 (0.5) T-20S138P446 ± 167 (186)>1000 (>416)>1000 (>416)>1000 (>416)Nucleophilic T-20S138T0.9 ± 0.2 (0.4)39 ± 8.5 (16)161 ± 35 (67)124 ± 43 (52)Aromatic T-20S138F9.4 ± 2.6 (4)203 ± 89 (85)393 ± 119 (164)478 ± 116 (200) T-20S138Y25 ± 9 (10)516 ± 223 (215)>1000 (>416)>1000 (>416) T-20S138V29 ± 14 (12)>1000 (>416)>1000 (>416)>1000 (>416)Amide T-20S138N19 ± 4 (8)>1000 (>416)>1000 (>416)>1000 (>416) T-20S138Q34 ± 11 (14)>1000 (>416)>1000 (>416)>1000 (>416)Acidic T-20S138D210 ± 94 (88)>1000 (>416)>1000 (>416)>1000 (>416) T-20S138E283 ± 80 (118)>1000 (>416)>1000 (>416)>1000 (>416)Basic T-20S138H210 ± 85 (88)>1000 (>416)>1000 (>416)>1000 (>416) T-20S138K708 ± 145 (295)>1000 (>416)>1000 (>416)>1000 (>416) T-20S138R362 ± 114 (150)>1000 (>416)>1000 (>416)>1000 (>416)a To improve the replication kinetics, D36G mutation, observed in the majority of HIV-1 strains, was introduced into the NL4-3 background used in this study (reference virus). Open table in a new tab Nucleophilic amino acid at position 138 of T-20 (T-20S138T) showed similar profiles. Conversely, aromatic and amide substitutions reduced the anti-HIV-1 activity of T-20 against HIV-1WT- and T-20-resistant clones. Other amino acid substitutions, especially acidic and basic amino acids, decreased the anti-HIV-1 inhibitory activity even against HIV-1WT. These results suggest that smaller hydrophobic (Ala > Leu, Ile) or more flexible (Met > Thr) residues are preferred in this position. Furthermore, the α-helical structure is important for the interaction, as a mutation to proline which is expected to disrupt the helix (25Nilsson I. Saaf A. Whitley P. Gafvelin G. Waller C. von Heijne G. J. Mol. Biol. 1998; 284: 1165-1175Crossref PubMed Scopus (114) Google Scholar) resulted in an inactive T-20 analog. Circular Dichroism—To clarify the mechanism by which the substitutions at Ser-138 influence the antiviral activity of T-20 derivatives, we examined the binding affinities of these peptides to N-HR using circular dichroism (CD) analysis (Fig. 2). CD spectra reveal the presence of stable α-helical structure of the 6-helix bundle that is a requisite for biological activity and is thought to be mechanistically and thermodynamically correlated with HIV-1 fusion (26Eckert D.M. Kim P.S. Annu. Rev. Biochem. 2001; 70: 777-810Crossref PubMed Scopus (1139) Google Scholar). Therefore, CD spectra typically at 222 nm indicate interaction of N-HR (N36) and C-HR (T-20 or C34). Because T-20 does not interact significantly in vitro with the N36 peptide, which is derived from amino acids 35–70 of N-HR, we used a derivative of C34, a peptide that overlaps with T-20 and also inhibits HIV fusion by the same mechanism. The C34 derivative contained the analogous T-20 substitutions described above (Fig. 1B). Consistent with antiviral activities, a mixture of N36 and C34S138P or C34S138W showed no apparent or reduced α-helicity, respectively. For binding with N36V38A or N36N43D, sufficient α-helicity at 25 °C was observed only in C34S138A, C34S138L, and C34S138T or C34S138A, C34S138L, and C34S138W, respectively (Fig. 2, A–C). To determine the thermal stability of the helical complexes formed from the N36 and C34 peptides, we measured the melting temperature (Tm) of each complex (supplemental Table 1). The sigmoidal transition of the CD signal at 222 nm correlates with the thermal stability of the helical complexes formed from the N36 and C34 peptides, which in turn are indicative of the binding affinity of these peptides. The melting temperature (Tm) indicating the 50% disruption of 6-helix bundle was comparatively evaluated. Complexes of N36 and C34 containing the S138A or S138L substitutions (N36/C34S138A or N36/C34S138L) showed high thermal stability, comparable with that of the wild-type N36/C34 complex. Similarly, the addition of the S138A or S138L also improved the thermal stability of the N36N43D/C34 complex. These results reveal a striking correlation between the thermal stability and the anti-HIV-1 activity of the complexes (R2 = 0.75, Fig. 3). The low Tm value of the complex formed from N36N43D and C34 suggests that virus containing the N43D mutation shows high resistance to T-20, likely due to less favorable thermodynamics that are expected to drive the formation of the 6-helix bundles containing T-20 inhibitor. Antiviral Activity of Substituted C34 at Ser-138—To confirm that binding of C34 to N-HR is indeed representative of T-20 binding to N-HR, we examined the anti-HIV-1 activities of C34-derived peptides that have S138A substitutions. The C34S138A and C34S138L peptides showed potent anti-HIV-1 activities, similar to T-20S138A and T-20S138L (supplemental Table 2). Based on these findings, we conclude that the stability of complexes comprised of modified C34s and N36s containing T-20 resistance mutations offers a good measure of the binding affinity of T-20S138X to N-HR. Antiviral Activity of C34 with N126K—We have recently identified another mutation at the N-HR of gp41 (N126K) during exposure of HIV-1 to C34 in vitro (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar). The N126K has been occasionally observed after prolonged T-20-containing therapy (10Cabrera C. Marfil S. Garcia E. Martinez-Picado J. Bonjoch A. Bofill M. Moreno S. Ribera E. Domingo P. Clotet B. Ruiz L. AIDS. 2006; 20: 2075-2080Crossref PubMed Scopus (40) Google Scholar, 15Xu L. Pozniak A. Wildfire A. Stanfield-Oakley S.A. Mosier S.M. Ratcliffe D. Workman J. Joall A. Myers R. Smit E. Cane P.A. Greenberg M.L. Pillay D. Antimicrob. Agents Chemother. 2005; 49: 1113-1119Crossref PubMed Scopus (155) Google Scholar). Here we have confirmed that the C34N126K peptide can also suppress a C34-resistant clone containing several mutations: I37K/N126K/L204I (Table 2). Therefore, peptides designed to have compensatory mutations seem to have potent antiviral activity. However, because residue 126 is located outside the amino acid sequence of T-20 (Fig. 1B), we could not examine the effect of N126K substitution on T-20 activity.TABLE 2Antiviral activity of C34N126K peptides against C34-resistant gp41 recombinant virusesEC50HIV-1WTaTo improve the replication kinetics, the D36G mutation, observed in majority of HIV-1 strains, was introduced into the NL4-3 background used in this study (reference virus).HIV-1ΔV4/I37K/N126K/L204IbC34-resistant HIV-1 was constructed with the reference virus as described (13). ΔV4 indicates 5 amino acids deletion (FNSTW) in the V4 region of gp120.nmC341.6 ± 0.35114 ± 29 (71)C34N126K0.95 ± 0.22 (0.6)1.1 ± 0.5 (0.7)a To improve the replication kinetics, the D36G mutation, observed in majority of HIV-1 strains, was introduced into the NL4-3 background used in this study (reference virus).b C34-resistant HIV-1 was constructed with the reference virus as described (13Nameki D. Kodama E. Ikeuchi M. Mabuchi N. Otaka A. Tamamura H. Ohno M. Fujii N. Matsuoka M. J. Virol. 2005; 79: 764-770Crossref PubMed Scopus (77) Google Scholar). ΔV4 indicates 5 amino acids deletion (FNSTW) in the V4 region of gp120. Open table in a new tab Replication Kinetics of Ser-138-substituted HIV-1—To evaluate the effect of Ser-138 substitutions on viral replication, we constructed molecular clones introducing several Ser-138 and determined their replication kinetics by measuring p24 gag antigen production in the culture supernatant. Single nucleotide changes to the TCA codon for Ser-138 may generate 4 amino acid substitutions, Ala, Thr, Leu, Pro, and Trp. As expected, the compensative substitution, S138A, in the T-20 resistance mutation N43D background enhanced replication kinetics of the N43D-containing clone as shown in supplemental Fig. 1. However, in the WT background the S138A appeared to decrease production of p24 as compared with HIV-1WT (Fig. 4). Other substitutions also reduced their replication kinetics. Interestingly, the S138W substitution did not show measurable p24 production. Syncytia induction and single cycle replication kinetics of the Ser-138-substituted HIV-1 were also examined (supplemental Fig. 2). Sizes of syncytia of each virus formed in the MAGI cells (supplemental Fig. 2, panels A–E) were associated with p24-normalized single-cycle infectivities (supplemental Fig. 2, panel F) and multicycle replication kinetics (Fig. 4). These results suggest that substitutions at Ser-138 are not likely to appear in the absence of T-20 therapy or the emergence of N43D mutation. Structure Modeling—The side chain of amino acid 138 (Ser or Ala) closely contacts with the hydrophobic pocket formed by Leu-44 and Leu-45 in the N-HR. The mutation from Ser to Ala increases hydrophobicity and may help to stabilize the N-HR/C-HR complex related with the potency of the HIV-1 fusion inhibitors (Fig. 5). Larger hydrophobic substitutions such as S138W, S138L, or S138I are likely to sterically interfere with efficient packing of the N-HR and C-HR helices. Similarly, introduction of charged residues at this region of the interface would also disrupt the hydrophobic environment and result in destabilized helix bundles, consistent with the biochemical and virological findings (Figs. 2, 3, 4 and Table 1). Based on crystallographic studies (27Bai X. Wilson K.L. Seedorf" @default.
• W2022901458 created "2016-06-24" @default.
• W2022901458 creator A5007153206 @default.
• W2022901458 creator A5014781635 @default.
• W2022901458 creator A5019527890 @default.
• W2022901458 creator A5026805844 @default.
• W2022901458 creator A5031384880 @default.
• W2022901458 creator A5033128839 @default.
• W2022901458 creator A5037427457 @default.
• W2022901458 creator A5039582262 @default.
• W2022901458 creator A5041513558 @default.
• W2022901458 creator A5063339600 @default.
• W2022901458 creator A5063947250 @default.
• W2022901458 creator A5065647772 @default.
• W2022901458 creator A5070368566 @default.
• W2022901458 creator A5073716313 @default.
• W2022901458 date "2009-02-01" @default.
• W2022901458 modified "2023-10-14" @default.
• W2022901458 title "Design of Peptide-based Inhibitors for Human Immunodeficiency Virus Type 1 Strains Resistant to T-20" @default.
• W2022901458 cites W1480555318 @default.
• W2022901458 cites W1659719610 @default.
• W2022901458 cites W1969338190 @default.
• W2022901458 cites W1973938190 @default.
• W2022901458 cites W1973984443 @default.
• W2022901458 cites W1991227629 @default.
• W2022901458 cites W1994081152 @default.
• W2022901458 cites W2000011855 @default.
• W2022901458 cites W2003658370 @default.
• W2022901458 cites W2003865428 @default.
• W2022901458 cites W2008845432 @default.
• W2022901458 cites W2027738518 @default.
• W2022901458 cites W2029346749 @default.
• W2022901458 cites W2030648131 @default.
• W2022901458 cites W2038063067 @default.
• W2022901458 cites W2040506631 @default.
• W2022901458 cites W2059023106 @default.
• W2022901458 cites W2085739552 @default.
• W2022901458 cites W2091847883 @default.
• W2022901458 cites W2100968031 @default.
• W2022901458 cites W2105517059 @default.
• W2022901458 cites W2111610276 @default.
• W2022901458 cites W2113396991 @default.
• W2022901458 cites W2118233996 @default.
• W2022901458 cites W2123797944 @default.
• W2022901458 cites W2124252851 @default.
• W2022901458 cites W2129160108 @default.
• W2022901458 cites W2133649749 @default.
• W2022901458 cites W2136097167 @default.
• W2022901458 cites W2141353064 @default.
• W2022901458 cites W2143312620 @default.
• W2022901458 cites W2152881035 @default.
• W2022901458 cites W2154681906 @default.
• W2022901458 cites W2158049965 @default.
• W2022901458 cites W4237410627 @default.
• W2022901458 doi "https://doi.org/10.1074/jbc.m807169200" @default.
• W2022901458 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2643509" @default.
• W2022901458 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19073606" @default.
• W2022901458 hasPublicationYear "2009" @default.
• W2022901458 type Work @default.
• W2022901458 sameAs 2022901458 @default.
• W2022901458 citedByCount "43" @default.
• W2022901458 countsByYear W20229014582012 @default.
• W2022901458 countsByYear W20229014582013 @default.
• W2022901458 countsByYear W20229014582014 @default.
• W2022901458 countsByYear W20229014582015 @default.
• W2022901458 countsByYear W20229014582017 @default.
• W2022901458 countsByYear W20229014582018 @default.
• W2022901458 countsByYear W20229014582020 @default.
• W2022901458 countsByYear W20229014582021 @default.
• W2022901458 countsByYear W20229014582022 @default.
• W2022901458 crossrefType "journal-article" @default.
• W2022901458 hasAuthorship W2022901458A5007153206 @default.
• W2022901458 hasAuthorship W2022901458A5014781635 @default.
• W2022901458 hasAuthorship W2022901458A5019527890 @default.
• W2022901458 hasAuthorship W2022901458A5026805844 @default.
• W2022901458 hasAuthorship W2022901458A5031384880 @default.
• W2022901458 hasAuthorship W2022901458A5033128839 @default.
• W2022901458 hasAuthorship W2022901458A5037427457 @default.
• W2022901458 hasAuthorship W2022901458A5039582262 @default.
• W2022901458 hasAuthorship W2022901458A5041513558 @default.
• W2022901458 hasAuthorship W2022901458A5063339600 @default.
• W2022901458 hasAuthorship W2022901458A5063947250 @default.
• W2022901458 hasAuthorship W2022901458A5065647772 @default.
• W2022901458 hasAuthorship W2022901458A5070368566 @default.
• W2022901458 hasAuthorship W2022901458A5073716313 @default.
• W2022901458 hasBestOaLocation W20229014581 @default.
• W2022901458 hasConcept C159047783 @default.
• W2022901458 hasConcept C2779281246 @default.
• W2022901458 hasConcept C3013748606 @default.
• W2022901458 hasConcept C55493867 @default.
• W2022901458 hasConcept C86803240 @default.
• W2022901458 hasConcept C89423630 @default.
• W2022901458 hasConceptScore W2022901458C159047783 @default.
• W2022901458 hasConceptScore W2022901458C2779281246 @default.
• W2022901458 hasConceptScore W2022901458C3013748606 @default.
• W2022901458 hasConceptScore W2022901458C55493867 @default.
• W2022901458 hasConceptScore W2022901458C86803240 @default.
• W2022901458 hasConceptScore W2022901458C89423630 @default.

• next