Bifunctional inhibition of HIV-1 reverse transcriptase: a first step in designing a bifunctional triphosphate.

The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof of concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T-6PEG-TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA.

Intravaginal ring delivery of the reverse transcriptase inhibitor TMC 120 as an HIV microbicide.

TMC 120 (Dapivirine) is a potent non-nucleoside reverse transcriptase inhibitor that is presently being developed as a vaginal HIV microbicide. To date, most vaginal microbicides under clinical investigation have been formulated as single-dose semi-solid gels, designed for application to the vagina before each act of intercourse. However, a clear rationale exists for providing long-term, controlled release of vaginal microbicides in order to afford continuous protection against heterosexually transmitted HIV infection and to improve user compliance. In this study we report on the incorporation of various pharmaceutical excipients into TMC 120 silicone, reservoir-type intravaginal rings (IVRs) in order to modify the controlled release characteristics of the microbicide. The results demonstrate that TMC 120 is released in zero-order fashion from the rings over a 28-day period and that release parameters could be modified by the inclusion of release-modifying excipients in the IVR. The hydrophobic liquid excipient isopropyl myristate had little effect on steady-state daily release rates, but did increase the magnitude and duration of burst release in proportion to excipient loading in the IVR. By comparison, the hydrophobic liquid poly(dimethylsiloxane) had little effect on TMC 120 release parameters. A hydrophilic excipient, lactose, had the surprising effect of decreasing TMC 120 burst release while increasing the apparent steady-state daily release in a concentration-dependent manner. Based on previous cell culture data and vaginal physiology, TMC120 is released from the various ring formulations in amounts potentially capable of maintaining a protective vaginal concentration. It is further predicted that the observed release rates may be maintained for at least a period of 1 year from a single ring device. TMC 120 release profiles and the mechanical properties of rings could be modified by the physicochemical nature of hydrophobic and hydrophilic excipients incorporated into the IVRs.

Structure of unliganded HIV-1 reverse transcriptase at 2.7 A resolution: implications of conformational changes for polymerization and inhibition mechanisms.

BACKGROUND: HIV-1 reverse transcriptase (RT) is a major target for anti-HIV drugs. A considerable amount of information about the structure of RT is available, both unliganded and in complex with template-primer or non-nucleoside RT inhibitors (NNRTIs). But significant conformational differences in the p66 polymerase domain among the unliganded structures have complicated the interpretation of these data, leading to different proposals for the mechanisms of polymerization and inhibition. RESULTS: We report the structure of an unliganded RT at 2.7 A resolution, crystallized in space group C2 with a crystal packing similar to that of the RT-NNRTI complexes. The p66 thumb subdomain is folded into the DNA-binding cleft. Comparison of the unliganded RT structures with the DNA-bound RT and the NNRTI-bound RT structures reveals that the p66 thumb subdomain can exhibit two different upright conformations. In the DNA-bound RT, the p66 thumb subdomain adopts an upright position that can be described as resulting from a rigid-body rotation of the p66 thumb along the "thumb's knuckle' located near residues Trp239 (in strand beta 14) and Val317 (in beta 15) compared with the thumb position in the unliganded RT structure. NNRTI binding induces an additional hinge movement of the p66 thumb near the thumb's knuckle, causing the p66 thumb to adopt a configuration that is even more extended than in the DNA-bound RT structure. CONCLUSIONS: The p66 thumb subdomain is extremely flexible. NNRTI binding induces both short-range and long-range structural distortions in several domains of RT, which are expected to alter the position and conformation of the template-primer. These changes may account for the inhibition of polymerization and the alteration of the cleavage specificity of RNase H by NNRTI binding.

Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain.

Reverse transcriptase (RT) serves as the replicative polymerase for retroviruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.

Bifunctional inhibition of human immunodeficiency virus type 1 reverse transcriptase: mechanism and proof-of-concept as a novel therapeutic design strategy.

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a major target for currently approved anti-HIV drugs. These drugs are divided into two classes: nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the synthesis and biochemical evaluation of a novel bifunctional RT inhibitor utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI) linked via a poly(ethylene glycol) (PEG) linker. HIV-1 RT successfully incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in a base-specific manner. Moreover, this inhibitor demonstrates low nanomolar potency that has 4.3-fold and 4300-fold enhancement of polymerization inhibition in vitro relative to the parent TMC-derivative and d4T, respectively. This study serves as a proof-of-concept for the development and optimization of bifunctional RT inhibitors as potent inhibitors of HIV-1 viral replication.

Structure and function of HIV-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition.

The rapid replication of HIV-1 and the errors made during viral replication cause the virus to evolve rapidly in patients, making the problems of vaccine development and drug therapy particularly challenging. In the absence of an effective vaccine, drugs are the only useful treatment. Anti-HIV drugs work; so far drug therapy has saved more than three million years of life. Unfortunately, HIV-1 develops resistance to all of the available drugs. Although a number of useful anti-HIV drugs have been approved for use in patients, the problems associated with drug toxicity and the development of resistance means that the search for new drugs is an ongoing process. The three viral enzymes, reverse transcriptase (RT), integrase (IN), and protease (PR) are all good drug targets. Two distinct types of RT inhibitors, both of which block the polymerase activity of RT, have been approved to treat HIV-1 infections, nucleoside analogs (NRTIs) and nonnucleosides (NNRTIs), and there are promising leads for compounds that either block the RNase H activity or block the polymerase in other ways. A better understanding of the structure and function(s) of RT and of the mechanism(s) of inhibition can be used to generate better drugs; in particular, drugs that are effective against the current drug-resistant strains of HIV-1.

Fidelity analysis of HIV-1 reverse transcriptase mutants with an altered amino-acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184.

Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV-1. The effects of this kind of substitution were determined in a lacZ-based assay using HIV-1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184-->Val and Glu89-->Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC-resistant Met184-->Val RT mutant an almost wild-type level of overall mutation frequency was observed, while the foscarnet-resistant RTs harbouring the Glu89-->Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183-->Phe mutant RT displayed a slightly lower fidelity than wild-type RT. Conversely, the ddI-resistant RT mutant containing the Leu74-->Val mutation showed a 3.5-fold higher fidelity compared to the wild-type enzyme. Finally, the Tyr115-->Ala substitution rendered the enzyme substantially more error-prone for DNA polymerization. These results correlate with three-dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV-1 RT.

Closing of the fingers domain generates motor forces in the HIV reverse transcriptase.

Using the force sensor of an atomic force microscope, motor forces of the human immunodeficiency virus-1 reverse transcriptase were measured during active replication of a short DNA transcript. At low load forces the polymerase is mechanically slowed, whereas at high force (approximately 15 piconewton) it stalls. From recordings of estimated polymerase turnover velocity versus load force, an approximate force-velocity curve has been constructed. The shape of the curve suggests that load force strongly inhibits the rate-limiting step of the polymerase turnover cycle and that the combined effect of load on all steps involves an effective motion of about 1.6 nm. Earlier results from pre-steady-state kinetics experiments have identified the rate-limiting step as the closing of the fingers domain to form a tight catalytic complex. Together these findings indicate that the closing of the fingers domain is a major force-generating step for human immunodeficiency virus reverse transcriptase and, by extension, for all DNA polymerase machines.

Single d(GpG)/cis-diammineplatinum(II) adduct-induced inhibition of DNA polymerization.

A 44 nucleotide DNA template containing a single site-specifically placed cisplatin adduct (cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]]) was annealed with a primer, positioning its 3'-end four bases before the adduct in the template strand. DNA polymerization in the presence of all four nucleotides revealed that both HIV-1 reverse transcriptase (RT) and T7 DNA polymerase strongly paused at one nucleotide preceding the first platinated guanine and at the positions opposite the two platinated guanines. Analysis of single nucleotide incorporation at each pause site showed that polymerization occurs with biphasic kinetics. A small percentage of DNA was bound productively, providing a small amplitude (1-3%) of a fast phase of polymerization, whereas most of the bound DNA (1-34%) was positioned at the pause site in a nonproductive manner and therefore elongated slowly (0.04-0.06 s-1). DNA substrates annealed to the cisplatin-modified template bind to HIV-1 RT with an affinity (10-20 nM) similar to that of unmodified substrates (6-9 nM). The cisplatin-DNA cross-link moderately weakened DNA binding to T7 DNA polymerase (12-115 nM) but significantly slowed the rate of incorporation of the next nucleotide (2-7 s-1 ), with larger effects closer to the cisplatin-DNA adduct. The crystal structure of the same cisplatin-DNA adduct [Takahara, P. M., Frederick, C. A., and Lippard, S. J. (1996) J. Am. Chem. Soc. 118, 12309-12321] reveals not only the bent DNA duplex but also the propeller twisted base pairs near the cisplatin-DNA adduct. The twisted base pairs may cause misalignment of the cisplatin-modified DNA at the binding cleft of T7 DNA polymerase and significantly slow the rate of the protein conformational change preceding polymerization, leading to the slight accumulation of intermediates within five base pairs of the adduct. The ground-state binding of the next correct nucleotide to the enzyme.DNA complex was weakened by the adduct with T7 DNA polymerase but unchanged with HIV-1 RT at sites other than the three strong pause sites. Nucleotide binding to both enzymes at the three strong pause sites was significantly weaker and less selective.