1. Characterization of the profiles of EFdA for potential clinical development.A number of individuals who received currently available nucleoside reverse transcriptase inhibitors (NRTIs) have developed resistant HIV-1 variants. However, there are only a few NRTIs that are on the pipeline as of today. The development of novel NRTIs, which are more potent and less toxic, and prevent or delay the emergence of resistant HIV-1 variants, is needed. We have previously identified 4?-ethynyl-substituted-2-halo-2?-deoxynucleosides, which potently inhibit the replication of various existing multi-NRTI-resistant HIV-variants in collaboration with scientists of Yamasa Corporation, Chiba, Japan. From such 4?-ethynyl congeners, we have chosen 4?-ethynyl-2-fluoro-2?-deoxyadenosine (EFdA) for further preclinical and clinical development, based on our findings on the potency and favorable pharmacokinetic/pharmacodynamic profiles potentially allowing once daily (QD) dosing schedule. In the period covered by this report, in order to ask the propensity of EFdA to allow the development of HIV-1 resistance to EFdA as compared to TDF, FTC, and another 4?-ethynyl NRTI, 4?-ethynyl-2?,3?-dideoxythymidine (Ed4T), we have started selection of HIVNL4-3 and a mixture of 11 multi-drug-resistant HIV-1 clinical isolates (HIV11MIX) as starting virus populations. We have already obtained a favorable resistance selection profile with EFdA, showing that the emergence of EFdA-resistant variants is significantly delayed compared to those of TDF and 4?-ethynyl-2?,3?-dideoxy-didehydro-thymidine (Ed4T) that is presently in early clinical trials. We also conducted pre-steady-state kinetics to examine the interactions of human mitochondrial DNA polymerase gamma with EFdA to assess the potential for EFdA toxicity in collaboration with Professor Anderson of Yale University. It was found that polymerase gamma incorporated EFdA-TP 4,300-fold less efficiently than its physiologic counterpart dATP, with an excision rate similar to ddATP. These data demonstrate that EFdA is a poor polymerase gamma substrate, suggesting minimal polymerase gamma-mediated toxicity of EFdA. In collaboration with Professor Michael Parniak of University of Pittsburgh, we administered EFdA (2 mg/kg, QD, subcutaneously) to 2 rhesus macaques that had shown end-stage SAIDS (chronic diarrhea, wasting, and persistent high plasma viral loads) at the time of initiation of EFdA treatment. EFdA showed excellent antiviral potency, bringing SIV viremia to undetectable level. SAIDS symptoms disappeared within 4 weeks of treatment and both macaques gained weight and showed no detectable adverse effects during 174 days on EFdA treatment. These data strongly suggest that EFdA is a promising candidate with a possibility of QD dosing schedule and less adverse effects than other NRTIs as a therapeutic for individuals harboring multidrug-resistant HIV-1 variants.2. Identification of novel protease inhibitors active against wild-type and multi-drug-resistant HIV-1 variants including DRV-resistant HIV-1 variants.We continued collaborative efforts with Professor Ghosh of Purdue University and identified more than 10 potent protease inhibitors and PDIs. Such novel agents include GRL-02031, GRL-216, GRL-0519, GRL-1398, and GRL-015-11 (Aoki, Ghosh, and Mitsuya: mansucript in preparation). Structural analysis was also performed for selected compounds using molecular modeling and x-ray crystallographic approaches.Analysis of crystal structure of a mutant HIV-1 protease isolated from a multi-drug-resistant clinical HIV-1 isolate (PRMDR-A02) in complex with GRL-0519 in comparison with crystal structures of APV and DRV with PRMDR-A02 was performed to possibly explain the greater potency of GRL-0519. We found that GRL-0519 is involved in maximum number of contacts with the MDR protease among the three compounds tested. Taken together, the bis-THF and tris-THF moieties as the P2 functional groups synergistically boost the potency of DRV and GRL-0519, respectively, explaining their greater inhibition profile compared to that of APV. Structure-function studies of two DRV analogs, one containing carboxybenzene (GRL007-11) and the other benzamide (GRL008-11) as the P2'moiety revealed that the GRL008-11 is at least >30-fold more potent against HIV-1NL4-3 than GRL007-11. GRL008-11 shows relatively greater number of overall contacts, explaining its greater potency.3. Study of the structure-activity relationship studies using various biological/antiviral assays and macromolecular crystallography. In this project, we used DRV structure as a lead template since it has a unique feature of direct polar contacts with the protease backbone, and new compounds were designed. Typically, the P2'aniline moiety of DRV was replaced with other moieties such as carboxybenzene and benzamide generating GRL-007-11 and GRL008-11, respectively. These two novel PIs were to be examined for their interactions with wild-type protease.Crystal structure of GRL-0519 in complex with PRMDR-A02 provided new insights regarding the binding and inhibition profiles of this novel compound. We co-crystallized various potent novel compounds in complex with PRMDR-A02 to better understand their potency. We also investigated the structural profiles of interactions of various novel PIs (DVR, TPV, and newly generated PIs etc.) with mutated protease molecules derived from various highly resistant HIV-1 variants such as DRV-resistant variants (HIV8MIXP51, HIVV32I/L33F/I54M/V82I, and HIVV32I/L33F/I54M/I84V), TPV-resistant HIV-1 variants (HIV11MIXP10, HIVL24M, HIVL33F, and HIVL33I), and DRV-resistant clinical HIV-1 variants.
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