Conformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs. pyrophosphorolysis – binding of foscarnet

HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3′-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg2+ ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg2+ (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.

Exploring the role of the α-carboxyphosphonate moiety in the HIV-RT activity of α-carboxy nucleoside phosphonates

As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the “L”-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the “L”-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2.

Scaling up strategies of the chronic respiratory disease programme of the European Innovation Partnership on Active and Healthy Ageing (Action Plan B3: Area 5)

Action Plan B3 of the European Innovation Partnership on Active and Healthy Ageing (EIP on AHA) focuses on the integrated care of chronic diseases. Area 5 (Care Pathways) was initiated using chronic respiratory diseases as a model. The chronic respiratory disease action plan includes (1) AIRWAYS integrated care pathways (ICPs), (2) the joint initiative between the Reference site MACVIA-LR (Contre les MAladies Chroniques pour un VIeillissement Actif) and ARIA (Allergic Rhinitis and its Impact on Asthma), (3) Commitments for Action to the European Innovation Partnership on Active and Healthy Ageing and the AIRWAYS ICPs network. It is deployed in collaboration with the World Health Organization Global Alliance against Chronic Respiratory Diseases (GARD). The European Innovation Partnership on Active and Healthy Ageing has proposed a 5-step framework for developing an individual scaling up strategy: (1) what to scale up: (1-a) databases of good practices, (1-b) assessment of viability of the scaling up of good practices, (1-c) classification of good practices for local replication and (2) how to scale up: (2-a) facilitating partnerships for scaling up, (2-b) implementation of key success factors and lessons learnt, including emerging technologies for individualised and predictive medicine. This strategy has already been applied to the chronic respiratory disease action plan of the European Innovation Partnership on Active and Healthy Ageing.