Mechanism-based Suppression of Dideoxynucleotide Resistance by K65R Human Immunodeficiency Virus Reverse Transcriptase Using an α-Boranophosphate Nucleoside Analogue

Abstract

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 observedin vivo. A decreased catalytic rate constantk 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. α-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. 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 observedin vivo. A decreased catalytic rate constantk 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. α-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. human immunodeficiency virus reverse transcriptase 3′-azido-3′-deoxythymidine 2′,3′-didehydro-2′,3′-dideoxythymidine (−)-β-l-2′,3′-dideoxy-3′-l-thiacytidine 3′-azido 3′-deoxythymidine 5′-monophosphate 2′,3′-didehydro-2′,3′-dideoxythymidine 5′-monophosphate 3′-amino 3′-deoxythymidine 5′-monophosphate (−)-β-l-2′,3′-dideoxy-3′-l-thiacytidine 5′-monophosphate 3′-azido-3′-deoxythymidine 5′-triphosphate 2′,3′-didehydro-2′,3′-dideoxythymidine 5′-triphosphate 3′-amino 3′-deoxythymidine 5′-triphosphate (−)-β-l-2′,3′-dideoxy-3′-l-thiacy-tidine 5′-triphosphate 2′-deoxynucleoside 5′-triphosphate 2′,3′-dideoxynucleoside 5′-triphosphate Nucleoside analogues are a major class of molecules active against the human immunodeficiency virus (HIV).1 Their viral target is reverse transcriptase (RT), an essential RNA- and DNA-dependent DNA polymerase encoded by the viralpol gene (1Lightfoote M.M. Coligan J.E. Folks T.M. Fauci A.S. Martin M.A. Venkatesan S. J. Virol. 1986; 60: 771-775Crossref PubMed Google Scholar). Nucleoside analogues must be phosphorylated to nucleoside 5′-triphosphate by cellular kinases in the infected cell to become efficient and specific viral DNA synthesis inhibitors. Most of the analogues do not possess a 3′-OH. Hence, when incorporated selectively into viral DNA by RT, DNA chain termination occurs and is responsible for the antiviral effect. During anti-retroviral therapies, the emergence of drug-resistant viruses limits the efficiency of nucleoside drugs, and this limitation represents a major cause of failure to control a HIV infection. Most drug-resistant viruses isolated from nucleoside analogue-treated patients harbor mutations in the viral pol gene (2Schinazi R. Larder B. Mellors J.W. Intl. Antiviral News. 2000; 8: 65-91Google Scholar). Remarkably, the set of resistance mutations selected during antiretroviral therapy is specific to the nucleoside drug. When 3′-azido-3′-deoxythymidine (AZT) is given as the sole drug, several patterns of amino acid changes involving M41L, D67N, K70R, L210W, T215F or Y, and K219Q give rise to up to a 200-fold AZT-resistant virus (3Larder B.A. Kemp S.D. Science. 1989; 246: 1155-1158Crossref PubMed Scopus (1016) Google Scholar, 4Larder B. Skalka A.M. Goff S.P. Reverse Transcriptase. CSHL Press, 1992: 205-222Google Scholar, 5Balzarini J. Biochem. Pharmacol. 1999; 58: 1-27Crossref PubMed Scopus (79) Google Scholar). When 2′,3′-dideoxynucleotides (ddNs) are given to patients, ddN-resistance emerges and is mainly correlated with single amino acid substitutions such as L74V or K65R (2Schinazi R. Larder B. Mellors J.W. Intl. Antiviral News. 2000; 8: 65-91Google Scholar, 5Balzarini J. Biochem. Pharmacol. 1999; 58: 1-27Crossref PubMed Scopus (79) Google Scholar, 6Gu Z. Fletcher R.S. Arts E.J. Wainberg M.A. Parniak M.A. J. Biol. Chem. 1994; 269: 28118-28122Abstract Full Text PDF PubMed Google Scholar, 7Shah F.S. Curr K.A. Hamburgh M.E. Parniak M. Mitsuya H. Arnez J.G. Prasad V.R. J. Biol. Chem. 2000; 275: 27037-27044Abstract Full Text Full Text PDF PubMed Google Scholar). The K65R mutation is found infrequently on viral isolates from patients treated with ddI and ddC (8Gu Z. Gao Q. Fang H. Salomon H. Parniak M.A. Goldberg E. Cameron J. Wainberg M.A. Antimicrob. Agents Chemother. 1994; 38: 275-281Crossref PubMed Scopus (203) Google Scholar, 9Zhang D. Caliendo A.M. Eron J.J. DeVore K.M. Kaplan J.C. Hirsch M.S. D'Aquila R.T. Antimicrob. Agents Chemother. 1994; 38: 282-287Crossref PubMed Scopus (144) Google Scholar). 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Agents Chemother. 1995; 39: 1888-1891Crossref PubMed Scopus (70) Google Scholar) or more processive (16Arion D. Borkow G. Gu Z. Wainberg M.A. Parniak M.A. J. Biol. Chem. 1996; 271: 19860-19864Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) DNA synthesis than wild-type RT, although the relationship between processivity and drug resistance is elusive. K65R RT shows resistance to ddNTPs relative to wild-type RT in steady-state kinetics assays by an unknown mechanism (6Gu Z. Fletcher R.S. Arts E.J. Wainberg M.A. Parniak M.A. J. Biol. Chem. 1994; 269: 28118-28122Abstract Full Text PDF PubMed Google Scholar, 17Gu Z. Arts E.J. Parniak M.A. Wainberg M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2760-2764Crossref PubMed Scopus (76) Google Scholar). The crystal structure of an RT·DNA·dNTP complex has revealed the catalytic mechanism of nucleotide incorporation into DNA (18Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar), but many questions remain concerning the role of K65R in drug resistance. Indeed, it can be seen in the crystal structure that the lysine 65 side chain contacts an on the of the nucleotide for at the reverse transcriptase active The of this to drug resistance is since the is from the position to could the of an incoming of lysine 65 and molecular of this substitution in the RT active suggested that K65R the of the ddNTP substrate D. H. Parniak M.A. Biochem. J. 2000; PubMed Google Scholar). However, since and ddNTPs are at it for 3′-OH is state kinetics is the of to the substrate efficiency of a nucleotide for The efficiency of incorporation of a nucleotide into DNA is given by the pol the rate constant for the creation of the phosphodiester and the constant of the nucleotide for RT K.A. Anderson J. Biol. Chem. Full Text PDF PubMed Google Scholar). of a ddNTP relative to its is by the efficiency of incorporation of the into DNA relative to that of the substrate this has been for various ddNTPs and wild-type RT, such not been for K65R this we of pre-steady state kinetics to the constant as as the catalytic constant pol of of the phosphodiester for several and ddNTP by either wild-type or K65R that the of an intramolecular between the 3′-OH and of the of the incoming nucleotide is for an efficient catalytic step. This intramolecular represents the to ddNTPs are up to better by K65R RT than wild-type that data can be used to design nucleotide analogues that drug resistance at the The wild-type RT gene has been J. S. J.M. B. J. B. Biochem. PubMed Scopus Google Scholar). This was and used to the RT gene at 65 and a in J. S. J.M. B. J. B. Biochem. PubMed Scopus Google were by and nucleotide were and as and active as J. S. J.M. B. J. B. Biochem. PubMed Scopus Google Scholar). DNA were from were and 2′,3′-dideoxynucleoside and were from 5′-triphosphate was and as A. B. J. Chem. 1998; PubMed Scopus Google Scholar, P. B. S. D. C. J. J. M. B. J. 2000; PubMed Scopus Google Scholar). are were by on a to in The has been B. J. J.M. J. S. C. S. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the used for both and assays was a DNA to a DNA The a single nucleotide for the nucleotide from the of the represents of the from DNA was by the of wild-type or K65R RT and of the and of at the of the of the nucleotide for at in RT The was by the of the nucleotide to a of in the of either or this the is in the of the nucleotide on of the found in the infected were during the of the and by The of is the of than than P. B. S. D. C. J. J. M. B. J. 2000; PubMed Scopus Google Scholar, B. J. J.M. J. S. C. S. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). state kinetics were and in with wild-type and K65R RT as K.A. Anderson J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of was with a A is the of the is the kinetic constant of of the phosphodiester bond, and is the kinetic constant of the The of on is by the and pol are the constant and the catalytic rate constant of the for RT, and pol were from The of RT in complex with DNA and nucleotide were from the The number is The crystal structure were and J. Google Scholar, A. C. Scholar). The K65R substitution in RT is responsible for viral resistance to a of the crystal structure of RT in complex with DNA and the group of lysine 65 contacts the of the incoming This phosphate is of the of the or of DNA are of drug resistance B. J. J.M. J. S. C. S. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P.R. A.M. 1999; 4: Full Text Full Text PDF PubMed Scopus Google Scholar), the K65R substitution might the and thus of the other K65R RT has been reported to against ddNTPs better than wild-type RT steady-state kinetic assays Z. Arts E.J. Parniak M.A. Wainberg M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2760-2764Crossref PubMed Scopus (76) Google Scholar, D. H. Parniak M.A. Biochem. J. 2000; PubMed Google Scholar). This that the K65R substitution is to either or with the 3′-OH of the incoming and that resistance by against ddNTPs than of a to which of might be in K65R ddNTP the crystal structure of a complex of RT, a DNA and a nucleotide was Examination of the crystal structure of the RT·DNA·dNTP complex (18Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar) shows that the 3′-OH of the incoming nucleotide an intramolecular with of the this the nucleotide is in the active as to the of the 3′-OH of the the of the incoming from lysine the chain of and the the The intramolecular not when a ddNTP is and this of ddNTPs are efficient for incorporation than dNTPs. This has been in both the DNA and the S. S. A.M. 1998; PubMed Scopus Google Scholar, H. A. J. 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Full Text Full Text PDF PubMed Scopus Google Scholar). this DNA synthesis by RT is at a specific position by incorporation of a in the of and either or are to the of the and synthesis of the of The of is of the of RT to the DNA chain or The are in RT is to DNA with efficiency when with other Using the efficiency of DNA is better than that observed the no on other The efficiency of the that the of the is for the agreement with by the DNA chain is by RT relative to DNA P.R. A.M. 1999; 4: Full Text Full Text PDF PubMed Scopus Google P.R. Schinazi R.F. Antimicrob. Agents Chemother. 2000; 44: PubMed Scopus Google Scholar). Using K65R RT, do not significant of DNA when with Using either K65R RT or wild-type RT, DNA is better than or DNA in the of DNA to level during the of the This that either the an between and synthesis during the of the or of the DNA complex is a which is for the in the of the could be a complex of RT, the and the nucleotide on of the P.R. A.M. 1999; 4: Full Text Full Text PDF PubMed Scopus Google Scholar, P.R. Schinazi R.F. Antimicrob. Agents Chemother. 2000; 44: PubMed Scopus Google Scholar). The was several of and RT no was a significant in the efficiency of of DNA observed between wild-type and K65R RT the level of of relative to DNA and the of wild-type relative to K65R RT that DNA is not in the mechanism of resistance by K65R of ddNTP at the RT active is a and was wild-type and K65R RT of a ddNTP relative to a can be pre-steady state kinetics of incorporation of a single nucleotide into DNA by RT K.A. Anderson J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.E. PubMed Scopus Google Scholar). the of a nucleotide for RT can be with the rate constant phosphodiester The is as the efficiency of nucleotide incorporation into and of incorporation between nucleotide were for and and the are in state kinetic of wild-type and K65R RT for DNA and pol were as were from and pol were as were from from and pol were as were from B. J. J.M. J. S. C. S. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in a new RT ddATP and relative to dATP and Using dATP and the from a of than from a in the catalytic rate Indeed, from to pol from to The incorporation from to the of a 3′-OH group in the the are when is with The of a 3′-OH group in the nucleotide the constant 2-fold to the catalytic constant to The incorporation from to the of a 3′-OH group in the that the catalytic rate constant pol at is than pol at a in agreement with K.A. Anderson J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.E. PubMed Scopus Google Scholar). that wild-type RT a resistance to ddATP and RT is to and ddATP and relative to dATP and The were K65R in this RT ddATP and relative to dATP and in a more pronounced than wild-type RT, Using dATP and the from a decreased than from a in the catalytic rate from to from to This an in incorporation from to the of a 3′-OH group in the both a in and a pol a in the efficiency of incorporation from to that K65R RT resistance to both ddATP and as by the and of ddATP and relative to dATP and to ddNTPs by a RT is as the between observed RT observed for wild-type K65R RT against ddATP wild-type RT against ddATP. K65R RT is resistant to ddATP. When are a of by K65R RT is with a by wild-type RT, a resistance to by K65R that in the of K65R RT resistance to ddATP at the of incorporation Indeed, and pol accounts for the efficiency as by a of from to This is the for a in pol from to is but the pol was by an in of for the K65R RT relative to wild-type This the efficiency of incorporation this in efficiency of incorporation was or the pre-steady state kinetics were and either wild-type or K65R The are in I. The K65R substitution the of RT for from to pol from to a 2-fold in catalytic efficiency K65R and that K65R has a on the catalytic rate constant and to a on the of the incoming that the catalytic of nucleotide incorporation into DNA is mostly in ddNTP resistance by K65R catalytic of the phosphodiester in intramolecular into the incoming nucleotide and not in the that this of the nucleotide could be used to design analogues an incorporation rate into this a on the nucleotide that the catalytic rate constant might be of for K65R RT since ddNTP resistance from a decreased rate constant relative to wild-type nucleotide analogues a the been P. B. S. D. C. J. J. M. B. J. 2000; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar). been found to be 2-fold better for RT than When in of either or the group DNA chain termination by wild-type and K65R RT P. B. S. D. C. J. J. M. B. J. 2000; PubMed Scopus Google Scholar). The position is in the incorporation were mainly to an catalytic it was of to this be the and K65R was and used in pre-steady state as The are in and the are in state kinetic of wild-type and K65R RT for and with dATP and for from were used to the relative to dATP or as for or for the is the of pol the is the pol pol the is the for dATP for for from were used to the relative to dATP or as for or for the is the of pol the is the pol pol the is the for dATP for and pol were as were and pol were as were from were used to the relative to dATP or as for or for the is the of pol the is the pol pol the is the for dATP for and pol were as were in a new Using wild-type RT, the of the group the incorporation of ddATP into This the in substrate efficiency between dATP and to leaving dATP as the substrate in the nucleotide Using K65R RT, the substrate in the of The is observed when ddATP is with and The of the group the incorporation efficiency of ddATP This represents a complete suppression of ddATP resistance by K65R the of the group is more pronounced on the catalytic rate constant pol than on the constant that ddNTP resistance by K65R RT is mainly to an catalytic rate constant pol for the incorporation of ddNTP into and that represents the to ddATP resistance by this drug resistance is a found during antiretroviral therapies, and is the of drug-resistant several of been at the molecular the RT has or either of the nucleotide or of the DNA chain J. Biochem. Pharmacol. 1999; 58: 1-27Crossref PubMed Scopus (79) Google D. Parniak M.A. Sci. 2000; PubMed Scopus Google Scholar). mechanism of resistance has been P.R. A.M. 1999; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). of the by AZT-resistant RT in a to the of the The of the phosphodiester to a 3′-OH and The of specific AZT-resistant mutations in RT has it the of a complex of RT, the DNA and the and it the catalytic of the mechanism P.R. A.M. 1999; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). Hence, is not at the AZT-resistant RT active relative to its but resistance from a wild-type RT, lysine 65 with the of the incoming The and of the become the leaving creation of the phosphodiester Hence, DNA synthesis has been by of a the of is the of to lysine this is a the K65R substitution might by K65R RT relative to wild-type that not the efficiency of of by wild-type and drug-resistant RT is relative to that of DNA but is not K65R RT relative to wild-type the K65R mechanism not a mechanism by either or such as in the of resistance. K65R has been as an AZT-resistant substitution H.Z. Hammond J.L. Cavalcanti S.C. Chu C.K. Schinazi R.F. Mellors J.W. Antimicrob. Agents Chemother. 2000; 44: 1783-1788Crossref PubMed Scopus (88) Google Scholar), that resistance and resistance do not a be of to the K65R substitution the catalytic rate constant of of the when in RT the AZT-resistant the of the is for efficient that the of the of the DNA is either of the or is in of phosphodiester a given wild-type level is to the of the between the and This is to the of the nucleotide that be by an amino acid substitution in it is to that during therapies, the of amino acid substitutions increasing ddNTP is The of the 3′-OH of the incoming nucleotide in has been for and DNA S. S. A.M. 1998; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar, J.W. K.A. 1996; PubMed Scopus Google Scholar). the complex of DNA and a it was that the group of could for the of the 3′-OH of the incoming DNA polymerase a the of DNA of the type DNA polymerase DNA and DNA is of an in E. DNA polymerase it of in reverse transcriptases and in shows that this intramolecular in the incoming nucleotide is for by the of in the of the 3′-OH and the to with the of DNA polymerase has a and can for the of other of the catalytic complex by a to a lower data that the of the the on the complex of and The of of the to ddNTP by K65R When is in resistance to amino acid substitutions or in the RT active to RT the to the nucleotide with a lower efficiency than its this lower efficiency of incorporation can from either a lower of the nucleotide for RT or a rate of incorporation of the nucleotide into DNA pol These are pre-steady state kinetics of incorporation of a single nucleotide into of this type of resistance mechanism is by the substitution in RT, which confers a resistance to in vivo and in vitro (2Schinazi R. Larder B. Mellors J.W. Intl. Antiviral News. 2000; 8: 65-91Google Scholar). RT the of for RT is relative to that for wild-type RT, the substitution the rate of incorporation into DNA R. U. R.S. PubMed Scopus Google Scholar). This substitution was the sole of drug resistance to nucleotide by a catalytic data that K65R is to a in the catalytic However, the substitution is to the and of the during (18Huang H. Chopra R. Verdine G.L. Harrison S.C. Science. 1998; 282: 1669-1675Crossref PubMed Scopus (1358) Google Scholar, R. U. R.S. PubMed Scopus Google Scholar), K65R is from the 3′-OH of the nucleotide from either or the molecular are The K65R substitution to the to the of between the 3′-OH and the of the incoming in the were used to analogues been found to the catalytic rate of phosphodiester P. B. S. D. C. J. J. M. B. J. 2000; PubMed Scopus Google Scholar). This is for K65R RT the 153-fold of this incorporation efficiency ddNTP resistance by this RT to be the of a nucleotide to target a RT and such catalytic rate that the of phosphodiester for incorporation into DNA is not a change K.A. Anderson J. Biol. Chem. Full Text PDF PubMed Google Scholar). is the mechanism for the rate to that the charge by the group at the position the for The of the group is for the charge on the and thus of the this from amino acid side of and which are for charge with nucleotide This is with the that the substitution not incorporation of but to a RT P. B. S. D. C. J. J. M. B. J. 2000; PubMed Scopus Google Scholar). that and no role in the for the a by the catalytic and The type of drug-resistant suppression might be of in the of other for which nucleotide resistance a decreased catalytic rate constant such as RT and resistance. The is a that might for RT, such as RT, that might a resistance as as for other nucleotide analogues such as and for which K65R is a resistance and for of the for and for help with