Biology subjects

Braz, J.

Publications and source records attributed to Braz, J..

3 recordsLinked to original sources

bioRxiv2023

Structure-Based Discovery of a NPFF1R Antagonist with Analgesic Activity

While opioid drugs remain among the most effective analgesics for pain management, adverse effects limit their use. Molecules that synergize with opioids, increasing analgesia without increasing side effects, could prove beneficial. A potential way to do so is via the RF-amide receptor system, as NPFFR1 agonists reduce {micro}- opioid receptor ({micro}OR)-based analgesia while antagonists increase it. These inferences are, however, clouded by the lack of selectivity of most NPFF1R ligands. Seeking selective antagonists of the NPFF1R, we screened a large virtual library against a homology model of NPFF1R. From 26 high-ranking molecules that were synthesized and tested, one antagonized NPFF1R with a Ki of 319 nM. Structure-based optimization led to a 22 nM antagonist of NPFF1R, compound 56, with selectivity against a large panel of GPCRs. When administered alone, 56 has no activity in mouse tail-flick nociception assays. However, coadministration of compound 56 and morphine produced significantly greater antinociception than did morphine alone, consistent with the notion that NPFF1R nociceptive activity occurs via modulation of {micro}OR signaling. Surprisingly, in the hot-plate assays 56 was analgesic by itself, suggesting that NPFF1R alone can also confer analgesia. At equi-analgesic doses, combinations of 56 with morphine reduced the common constipation side effect of morphine versus using morphine alone. The high selectivity of 56 and its activity in cooperation with morphine supports further analgesic development against NPFF1R and against the RF-amide family of receptors more generally.

pharmacology and toxicology↗

bioRxiv2023

Tetracycline transactivator overexpression in keratinocytes triggers a TRPV1 primary sensory neuron-dependent neuropathic itch

Mouse models that combine tetracycline-controlled gene expression systems and conditional genetic activation can tightly regulate transgene expression in discrete cell types and tissues. However, the commonly used Tet-Off variant, tetracycline transactivator (tTA), when overexpressed and fully active, can lead to developmental lethality, disease, or more subtle behavioral phenotypes. Here we describe a profound itch phenotype in mice expressing a genetically encoded tTA that is conditionally activated within the Phox2a lineage. Phox2a; tTA mice develop intense, localized scratching and regional skin lesions that can be controlled by the tTA inhibitor, doxycycline. As gabapentin, but not morphine, relieved the scratching, we consider this phenotype to result from chronic neuropathic itch, not pain. In contrast to the Phox2a lineage, mice with tTA activated within the Phox2b lineage, which has many similar areas of recombination within the nervous system, did not recapitulate the scratching phenotype. In Phox2a-Cre mice, but not Phox2b-Cre, intense Cre-dependent reporter expression was found in skin keratinocytes which formed the area at which skin lesions developed. Most interestingly, repeated topical application of the DREADD agonist, CNO, which chronically induced Gi signaling in Phox2a-keratinocytes, completely reversed the localized scratching and skin lesions. Furthermore, ablation of TRPV1-expressing, primary afferent neurons reduced the scratching with a time course comparable to that produced by Gi-DREADD inhibition. These temporal properties suggest that the neuropathic itch condition arises not only from localized keratinocyte activation of peripheral nerves but also from a persistent, gabapentin-sensitive state of central sensitization.

neuroscience↗

bioRxiv2022

Structure-based Discovery of Conformationally Selective Inhibitors of the Serotonin Transporter

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has an unusual anti-depressant profile and stabilizes the inward-open conformation. Unfortunately, ibogaine is promiscuous and cardiotoxic, limiting understanding of inward-open state ligands. We computationally docked over 200 million small molecules against the ibogaine stabilized inward-open state of SERT. Thirty-six top-ranking compounds were synthesized and thirteen inhibited with potencies ranging from 29 to 5000 nM. Structure-based optimization led to two novel inhibitors with Ki values down to 3 nM. The new molecules stabilized an outward-closed state of the transporter and had little activity against off-targets. A cryo-EM structure of one of these bound to SERT confirmed the predicted geometry. In mouse behavioral assays, both had anxiolytic and anti-depressant activity, with potencies up to 200 better than fluoxetine.

biochemistry↗