Biology subjects

Hanak, F.

Publications and source records attributed to Hanak, F..

3 recordsLinked to original sources

bioRxiv2025

Exploiting the Angiotensin-Converting Enzyme Pathway to Augment Endogenous Opioid Signaling

Angiotensin Converting Enzyme (ACE) impacts hemodynamics by regulating the conversion of angiotensin I to the vasoconstricting angiotensin II. We recently identified a non-canonical central role of ACE in the degradation of enkephalin heptapeptide, Met-enkephalin-Arg-Phe (MERF). Enkephalins are short-lived, endogenous opioid peptides that mediate the bodys intrinsic analgesic response. Here we identify chemically diverse ACE inhibitors using an optimized high throughput screening assay to boost endogenous opioid signaling. Our primary hits (thiorphan, D609, and raloxifene) were selected for dose-response characterization, in vitro enkephalin release, in vivo analgesic potency, and in silico analysis. Intracerebroventricular administration of these compounds significantly attenuated pain response, alone and in combination with MERF, which was reversed by opioid receptor antagonist naloxone. Molecular docking provided additional insight into the active site interactions of these scaffolds, which could be exploited further for creation of more potent inhibitors. These results showcase the potential of central ACE inhibitors to modulate endogenous MERF signalling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/639161v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1b8ce53org.highwire.dtl.DTLVardef@1f1cbd3org.highwire.dtl.DTLVardef@17cca3eorg.highwire.dtl.DTLVardef@1c1b35c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Inhibition of the angiotensin-converting enzyme N-terminal catalytic domain prevents endogenous opioid degradation in brain tissue

Angiotensin-converting enzyme (ACE) regulates blood pressure by cleaving angiotensin peptides in the periphery, and can also regulate endogenous opioid signaling by degrading enkephalin peptides in the brain. ACE has two catalytic domains, located in the N-terminal or C-terminal region of the protein, but little is known about the roles that these two catalytic domains play in regulating endogenous opioid degradation in brain tissue. Using acute brain slice preparations from mice of both sexes, we developed methods to study degradation of Met-enkephalin-Arg-Phe (MERF) by ACE, and investigated the role of each ACE catalytic domain in MERF degradation. Using mutant mouse lines with functional inactivation of either the N-terminal domain or the C-terminal domain, we incubated acute brain slices with exogenous MERF at a saturating concentration. The degradation MERF to produce Metenkephalin was only reduced by N-terminal domain inactivation. Additionally, application of a selective N-terminal domain inhibitor (RXP407) reduced degradation of both exogenously applied and endogenously released MERF, while a selective C-terminal domain inhibitor (RXPA380) had no effect. Taken together, our results suggest that the ACE N-terminal domain is the primary site of MERF degradation in brain tissue, and that N-terminal domain inhibition is sufficient to reduce degradation of this specific endogenous opioid peptide. Our results have exciting implications for the development of novel pharmacotherapies that target the endogenous opioid system to treat psychiatric and neurological disorders.

neuroscience↗

bioRxiv2024

Modulation of endogenous opioid signaling by inhibitors of puromycin sensitive aminopeptidase

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.

pharmacology and toxicology↗