The Stereoselective Targeting of a Specific Enzyme-Substrate Complex Is the Molecular Mechanism for the Synergic Inhibition of HIV-1 Reverse Transcriptase by (R)-(−)-PPO464

Abstract

The human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitor pyrrolopyridooxazepinone (PPO) derivative, (؎)-PPO294, was shown to be active toward wild type and mutated HIV-1 RT and to act synergistically in combination with 3-azido-3-deoxythymidine (Campiani, G., Morelli, E., Fabbrini, M., Nacci, V., Greco, G., Novellino, E., Ramunno, A., Maga, G., Spadari, S., Caliendo, G., Bergamini, A., Faggioli, E., Uccella, I., Bolacchi, F., Marini, S., (1999) J. Med. Chem. 42, 4462-4470). The (؎)-PPO294 racemate was resolved into its pure enantiomers, and the absolute configuration was determined by x-ray analysis. The virus-encoded HIV-1 1 reverse transcriptase (RT) is essential for the viral replication cycle and therefore represents a logical target for antiviral chemotherapy (1, 2). Recently, a class of inhibitors targeted to the viral RT, the so-called NNRTIs, have gained a definitive place in the treatment of HIV-1 infections along with NRTIs and PIs (3). These compounds, despite their different chemical structures, are highly specific for HIV-1 RT and bind to the enzyme at the same allosteric site close to, but distinct from, the catalytic site, behaving as typically noncompetitive inhibitors with respect to the different substrates of the polymerization reaction. The NNRTIs nevirapine, delavirdine, and the most recently licensed, efavirenz (Fig. 1), are currently used in clinical practice. First generation NNRTIs, such as nevirapine, were identified by extensive random screening of different molecules. However, given their very similar mode of action and their unique binding site in the RT enzyme, the occurrence of just a few single amino acid substitutions in the RT gene can confer resistance to most of the first generation NNRTIs such as nevirapine (4). Accumulating knowledge about the resistance mutations selected by these compounds during chemotherapy as well as the resolution of crystallographic structures of different complexes of HIV-1 RT bound to NNRTIs (5, 6) allowed the rational design of second generation molecules, such as efavirenz, with improved potency against the wild type enzyme and several resistant forms (7). However, the single K103N mutation can still confer crossresistance to most, if not all, of the members of this subclass (8). Thus, the continuous challenge posed by the emergence of HIV-1-resistant mutants highlights the need to develop third generation inhibitors with yet more favorable binding properties to the wild type and mutated enzymes (9).Triple drug combinations have been shown to suppress plasma HIV-1 load for periods of time significantly longer than monotherapy or dual therapy, allowing CD4T cell increase, * This work was supported in part by ISS-Programma Nazionale di Ricerca sull' AIDS Contract 30C.70 (to S. S. and G. M.), 30C.34 (to F. B., S. P., and M. Z.), and 40C.65 (to V. N. and G. C.); by the Consiglio Nazionale delle Ricerche Target Project on Biotechnology (to S. S.); by Universita' di Siena Grant PARR 99 (to G. C.); and by contrac...

Effects of Drug Resistance Mutations L100I and V106A on the Binding of Pyrrolobenzoxazepinone Nonnucleoside Inhibitors to the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Catalytic Complex

“…Previous observations indicated that the inhibition of HIV-1 RT by the NNRTI PPO464 was due to the specific binding of this molecule to the ternary complex formed by RT, the template primer, and the dNTP (25). In the present study we extended the analysis to molecules belonging to a related class of NNRTIs, the PBO derivatives, whose structures are shown in Fig.…”

“…In addition, we have shown that the lead compound of this class, (R)-(Ϫ)-PPO464, was selectively targeting the ternary complex formed by the viral RT with its substrates nucleic acid and nucleotide. It showed a significant synergy with the NRTI zidovudine and a favorable pharmacokinetic profile in mice and rats, either alone or during coadministration with the HIV-1 PI ritonavir (25). The results of the biological and pharmacokinetic experiments suggest a potential clinical utility of PBO and PPO analogs in combination with NRTIs against strains of HIV-1 bearing mutations that confer resistance to known NNRTIs.…”

“…We have previously shown that the NNRTI (R)-(Ϫ)-PPO464 specifically targets the catalytically competent ternary complex of RT with its nucleic acid and nucleotide substrates (25). As a consequence, the potency of inhibition of (R)-(Ϫ)-PPO464 was shown to increase as the concentration of the substrates was increased.…”

High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

“…This means that, in principle, the NNBS might not be identical in these three mechanistic forms. Several kinetic studies have shown that this is indeed the case, so that some NNRTIs selectively target one or a few of the different enzymatic forms along the reaction pathway (5,13,15). This observation likely reflects the different spatial rearrangements not only of the NNBS itself but also of the adjacent nucleotide binding site (3,20,26,27).…”

“…The ethyl ester of N-{3-[ (3,5- Nucleic acid substrates. The homopolymer poly(rA) (Pharmacia) was mixed at weight ratios in nucleotides of 10:1 to the oligomer oligo(dT) [12][13][14][15][16][17][18] (Pharmacia) in 20 mM Tris-HCl (pH 8.0) containing 20 mM KCl and 1 mM EDTA, heated at 65°C for 5 min, and then slowly cooled at room temperature.…”