Imidazole Bioisostere Activators of Endopeptidase Neurolysin with Enhanced Potency and Metabolic Stability.

The peptidase neurolysin (Nln) has been validated as a potential target for developing therapeutics for ischemic stroke (IS). Overexpression of Nln in a mouse model of IS provides significant cerebroprotection, leading to reduced infarction size and edema volume. Pharmacological inhibition of Nln in the post-stroke brain worsens neurological outcomes. A virtual screen identified dipeptide small-molecule activators of Nln. Optimization studies resulted in a class of peptidomimetic compounds with promising activity. However, these compounds still possessed an amide bond that compromised their stability in plasma and the brain. Herein, we report the synthesis and characterization of a series of amide bioisosteres based on our peptidomimetic leads. Imidazole-based bioisosteres afford scaffolds with increased potency to activate Nln combined with enhanced mouse plasma stability and significantly better brain permeability over the original dipeptide hits.

Structure-activity relationship studies of functionalized aromatic peptidomimetics as neurolysin activators.

Modulating peptidase neurolysin (Nln) has been identified as a potential cerebroprotective target for the development of therapeutics for ischemic stroke. Continued structure-activity relationship studies on peptidomimetic small molecule activators of Nln bearing electron-donating and electron- withdrawing functionalized phenyls are explored. Incorporation of fluorine or trifluoromethyl groups produces Nln activators with enhanced A50, while methoxy substitution produces derivatives with enhanced Amax. Selected activators containing methoxy or trifluoromethyl substitution are selective for Nln over related peptidases and possess increased blood-brain barrier penetrability than initial hits.

In-Vivo and Ex-Vivo Brain Uptake Studies of Peptidomimetic Neurolysin Activators in Healthy and Stroke Animals.

PURPOSE: Neurolysin (Nln) is a peptidase that functions to preserve the brain following ischemic stroke by hydrolyzing various neuropeptides. Nln activation has emerged as an attractive drug discovery target for treatment of ischemic stroke. Among first-in-class peptidomimetic Nln activators, we selected three lead compounds (9d, 10c, 11a) for quantitative pharmacokinetic analysis to provide valuable information for subsequent preclinical development. METHODS: Pharmacokinetic profile of these compounds was studied in healthy and ischemic stroke-induced mice after bolus intravenous administration. Brain concentration and brain uptake clearance (Kin) was calculated from single time point analysis. The inter-relationship between LogP with in-vitro and in-vivo permeability was studied to determine CNS penetration. Brain slice uptake method was used to study tissue binding, whereas P-gp-mediated transport was evaluated to understand the potential brain efflux of these compounds. RESULTS: According to calculated parameters, all three compounds showed a detectable amount in the brain after intravenous administration at 4 mg/kg; however, 11a had the highest brain concentration and brain uptake clearance. A strong correlation was documented between in-vitro and in-vivo permeability data. The efflux ratio of 10c was ~6-fold higher compared to 11a and correlated well with its lower Kin value. In experimental stroke animals, the Kin of 11a was significantly higher in ischemic vs. contralateral and intact hemispheres, though it remained below its A50 value required to activate Nln. CONCLUSIONS: Collectively, these preclinical pharmacokinetic studies reveal promising BBB permeability of 11a and indicate that it can serve as an excellent lead for developing improved drug-like Nln activators.

Discovery of First-in-Class Peptidomimetic Neurolysin Activators Possessing Enhanced Brain Penetration and Stability.

Peptidase neurolysin (Nln) is an enzyme that functions to cleave various neuropeptides. Upregulation of Nln after stroke has identified the enzyme as a critical endogenous cerebroprotective mechanism and validated target for the treatment of ischemic stroke. Overexpression of Nln in a mouse model of stroke results in dramatic improvement of stroke outcomes, while pharmacological inhibition aggravates them. Activation of Nln has therefore emerged as an intriguing target for drug discovery efforts for ischemic stroke. Herein, we report the discovery and hit-to-lead optimization of first-in-class Nln activators based on histidine-containing dipeptide hits identified from a virtual screen. Adopting a peptidomimetic approach provided lead compounds that retain the pharmacophoric histidine moiety and possess single-digit micromolar potency over 40-fold greater than the hit scaffolds. These compounds exhibit 5-fold increased brain penetration, significant selectivity over highly homologous peptidases, greater than 65-fold increase in mouse brain stability, and 'drug-like' fraction unbound in the brain.