Vo, S. V. | bioRxiv Science

bioRxiv2025

AMPA receptor activation within the prelimbic cortex is necessary for incubated cocaine-craving

The incubation of craving is a behavioral phenomenon in which cue-elicited craving increases during a period of drug abstinence. Incubated cocaine-craving is associated with increased extracellular glutamate within the medial prefrontal cortex (mPFC) and this release, particularly within the prelimbic (PL) subregion, is necessary for incubated cocaine-craving. A potential candidate mediating these incubation-driving effects of glutamate release within the PL are alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). To investigate the role of mPFC AMPARs in incubated craving, male and female Sprague-Dawley rats were trained to self-administer cocaine for 6 h/day for 10 consecutive days. Either during early or later withdrawal, rats were infused intra-PL with the AMPAR antagonist NBQX (0 or 1 {micro}g/0.5 {micro}l per side), followed by 30-min tests for cue-reinforced responding. Immunoblotting was also conducted to relate the expression of incubated cocaine- and sucrose-craving to AMPAR subunit expression within mPFC subregions. Intra-PL NBQX blocked incubated craving expressed in late, but not early, withdrawal. No incubation-related changes in AMPAR subunit expression were detected within the PL or IL of rats of either sex and no estrus-associated changes in subunit expression were detected in female rats exhibiting incubated cocaine-craving. In contrast, elevated GluA1 expression was observed within the IL of male rats exhibiting an incubation of sucrose-craving. Together, these findings indicate a necessary role for AMPARs within the PL in driving incubated cocaine-craving and suggest that AMPARs located within the IL may be involved also in sucrose-craving selectively in males.

neuroscience↗

bioRxiv2021

Deciphering the Molecular Mechanism of HCV Protease Inhibitor Fluorination as a General Approach to Avoid Drug Resistance

Third generation Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs), glecaprevir and voxilaprevir, are highly effective across genotypes and against many resistant variants. Unlike earlier PIs, these compounds have fluorine substitutions on the P2-P4 macrocycle and P1 moieties. Fluorination has long been used in medicinal chemistry as a strategy to improve physicochemical properties and potency. However, the molecular basis by which fluorination improves potency and resistance profile of HCV NS3/4A PIs is not well understood. To systematically analyze the contribution of fluorine substitutions to inhibitor potency and resistance profile, we used a multi-disciplinary approach involving inhibitor design and synthesis, enzyme inhibition assays, co-crystallography, and structural analysis. A panel of inhibitors in matched pairs were designed with and without P4 cap fluorination, tested against WT protease and the D168A resistant variant, and a total of 22 high-resolution co-crystal structures were determined. While fluorination did not significantly improve potency against the WT protease, PIs with fluorinated P4 caps retained much better potency against the D168A protease variant. Detailed analysis of the co-crystal structures revealed that PIs with fluorinated P4 caps can sample alternate binding conformations that enable adapting to structural changes induced by the D168A substitution. Our results elucidate molecular mechanisms of fluorine-specific inhibitor interactions that can be leveraged in avoiding drug resistance.

biochemistry↗