Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

Abstract

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.

Structure-guided approach identifies a novel class of HIV-1 ribonuclease H inhibitors: binding mode insights through magnesium complexation and site-directed mutagenesis studies

“…5,6 The number of newly infected people/year is constant and among them there is an overall increase of transmitted drug resistance mutations (TDRMs) detected in antiretroviral treatment-naive patients. 2,7 The resistance, which often results from lack of compliance, can relay to multiple types of drug, and can dramatically affect the outcome of HAART, 8,9 therefore, there is a strong need to search for non-traditional chemo-targets for anti-HIV drug developments. [10][11][12] Inhibition of the HIV-1 reverse transcriptase associated ribonuclease H (RNase H) function provides a novel target for anti-HIV chemotherapy, since it is the only viral encoded enzymatic function for which no inhibitor went in clinical development so far.…”

Novel natural non-nucleoside inhibitors of HIV-1 reverse transcriptase identified by shape- and structure-based virtual screening techniques

“…6 Despite the remarkable successes of current therapies, antiviral drug resistance severely limits the efficacy of long-term treatment regimens due to continuous viral replication and prolonged exposure to the drugs, thus impairing the effectiveness of the combined drugs. 3 The successive selection of additional mutations can lead to multidrug resistance that dramatically affects the outcome of HAART, 7 enlightening the need of a constant effort to identify novel inhibitors. [8][9][10] Additionally, new antiretroviral agents are under development to attempt to improve the pharmaceutical properties of the drugs, with the aim to achieve better dosing profiles, minimize side effect profiles and increase the efficacy.…”

Prenylated phloroglucinols from Hypericum scruglii, an endemic species of Sardinia (Italy), as new dual HIV-1 inhibitors effective on HIV-1 replication

“…Although the global scale-up of antiretroviral therapy has contributed to reduce the number of new infections and AIDS-related deaths, about 37 million people were estimated to be infected with HIV in 2016, with 1.8 million of new infections and 1 million of deaths [ 19 ]. To date there is no vaccine or cure for HIV infection, and the efficacy of antiretroviral therapy, which combines two or three antiviral agents, targeting different steps of the virus replication cycle, can be compromised by the selection of strains resistant to one or multiple drug classes [ 20 , 21 ] and treatment-associated toxicity [ 22 ], requiring the discovery of new antiviral agents with innovative modes of action or targets. In this respect, the identification of one molecule able to inhibit more than one viral function would provide significant advantages, raising the genetic barrier to resistance and increasing the compliance to therapy.…”