Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)-derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium (RPE) in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease (GVHD), and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases.

We have designed a nitroaromatic photochemical protecting group that absorbs visible light in the violet-blue range. The chromophore is a dinitro derivative of bisstyrylthiophene (or BIST) that absorbs light very effectively (ε440 = 66,000 M−1 cm−1 and two-photon cross section of 350 GM at 775 nm). We developed a “caged calcium” molecule by conjugation of BIST to a Ca2+ chelator that upon laser flash photolysis rapidly releases Ca2+ in less than 0.2 ms. Using the patch-clamp method the optical probe, loaded with Ca2+, was delivered into acutely isolated mouse cardiac myocytes, where either one- and two-photon uncaging of Ca2+ induced highly local or cell-wide physiological Ca2+ signaling events.

Caged compounds have widely used by neurophysiologists to study many aspects of cellular signaling in glia and neurons. Biologically inert before irradiation, they can be loaded into cells via patch pipette or topically applied in situ to a defined concentration, photolysis releases the caged compound in a very rapid and spatially defined way. Since caged compounds are exogenous optical probes, they include not only natural products such neurotransmitters, calcium and IP3, but non-natural products such as fluorophores, drugs and antibodies. In this Technical Spotlight we provide a short introduction to the uncaging technique by discussing the nitroaromatic caging chromophores most widely used in such experiments (e.g. CNB1, DMNB, MNI and CDNI). We show that recently developed caging chromophores (RuBi and DEAC450) that are photolyzed with blue light (ca. 430–480 nm range) can be combined with traditional nitroaromatic caged compounds to enable two-color optical probing of neuronal function. For example, one-photon uncaging of either RuBi-GABA or DEAC450-GABA with a 473-nm laser is facile, and can block non-linear currents (dendritic spikes or action potentials) evoked by two-photon uncaging of CDNI-Glu at 720 nm. We also show that two-photon uncaging of DEAC450-Glu and CDNI-GABA at 900 and 720 nm, respectively, can be used to fire and block action potentials. Our experiments illustrate that recently developed chromophores have taken uncaging out of the “monochrome era”, in which it has existed since 1978, so as to enable multichromic interrogation of neuronal function with single synapse precision.

A number of fatty acyl derivatives of (-)-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC, 1) were synthesized and evaluated for their anti-HIV activity. The monosubstituted 5'-O-fatty acyl derivatives of 3TC (EC(50) = 0.2-2.3 μM) were more potent than the corresponding monosubstituted N(4)-fatty acyl (EC(50) = 0.4-29.4 μM) and 5'-O-N(4)-disubstituted (EC(50) = 72.6 to >154.0 μM) derivatives of the nucleoside. 5'-O-Myristoyl (16) and 5'-O-12-azidododecanoyl derivatives (17) were found to be the most potent compounds (EC(50) = 0.2-0.9 μM) exhibiting at least 16-36-fold higher anti-HIV activity against cell-free virus than 1 (EC(50) = 11.4-32.7 μM). The EC(90) values for 16 against B-subtype and C-subtype clinical isolates were several folds lower than those of 1. The cellular uptake studies confirmed that compound 16 accumulated intracellularly after 1 h of incubation with CCRF-CEM cells and underwent intracellular hydrolysis. 5'-O-Fatty acyl derivatives of 1 showed significantly higher anti-HIV activity than the corresponding physical mixtures against the B-subtype virus.

Three fatty acyl conjugates of (-)-2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC, emtricitabine) were synthesized and evaluated against HIV-1 cell-free and cell-associated virus and compared with the corresponding parent nucleoside and physical mixtures of FTC and fatty acids. Among all the compounds, the myristoylated conjugate of FTC (5, EC(50) = 0.07-3.7 μM) displayed the highest potency. Compound 5 exhibited 10-24 and 3-13-times higher anti-HIV activity than FTC alone (EC(50) = 0.7-88.6 μM) and the corresponding physical mixtures of FTC and myristic acid (14, EC(50) = 0.2-20 μM), respectively. Cellular uptake studies confirmed that compound 5 accumulated intracellularly after 1 h of incubation and underwent intracellular hydrolysis in CCRF-CEM cells. Alternative studies were conducted using the carboxyfluorescein conjugated with FTC though β-alanine (12) and 12-aminododecanoic acid (13). Acylation of FTC with a long-chain fatty acid in 13 improved its cellular uptake by 8.5-20 fold in comparison to 12 with a short-chain β-alanine. Compound 5 (IC(90) = 15.7-16.1 nM) showed 6.6- and 35.2 times higher activity than FTC (IC(90) = 103-567 nM) against multidrug resistant viruses B-NNRTI and B-K65R, indicating that FTC conjugation with myristic acid generates a more potent analogue with a better resistance profile than its parent compound.