Evaluation of Systemic and Brain Pharmacokinetic Parameters for Repurposing Metformin Using Intravenous Bolus Administration.

Metformin's potential in treating ischemic stroke and neurodegenerative conditions is of growing interest. Yet, the absence of established systemic and brain pharmacokinetic (PK) parameters at relevant pre-clinical doses presents a significant knowledge gap. This study highlights these PK parameters and the importance of using pharmacologically relevant pre-clinical doses to study pharmacodynamics (PD) in stroke and related neurodegenerative diseases. An LC-MS/MS method to measure metformin levels in plasma, brain, and cerebrospinal fluid (CSF) was developed and validated. In vitro assays examined brain tissue binding and metabolic stability. Intravenous (IV) bolus administration of metformin to C57BL6 mice covered low to high dose range maintaining pharmacological relevance. Quantification of metformin in the brain was used to assess brain pharmacokinetic parameters, such as unidirectional blood-to-brain constant (Kin) and unbound brain-to-plasma ratio (Kp, uu, brain). Metformin exhibited no binding in the mouse plasma and brain and remained metabolically stable. It rapidly entered the brain, reaching detectable levels in as little as 5 minutes. A Kin value of 1.87 {plus minus} 0.27 µl/g/min was obtained. As the dose increased, Kp, uu, brain showed decreased value, implying saturation, but this did not affect an increase in absolute brain concentrations. Metformin was quantifiable in the CSF at 30 minutes but decreased over time, with concentrations lower than those in the brain across all doses. Our findings emphasize the importance of metformin dose selection based on pharmacokinetic parameters for pre-clinical pharmacological studies. We anticipate further investigations focusing on pharmacokinetics and pharmacodynamics (PKPD) in disease conditions, such as stroke. Significance Statement The study establishes crucial pharmacokinetic parameters of metformin for treating ischemic stroke and neurodegenerative diseases, addressing a significant knowledge gap. It further emphasizes the importance of selecting pharmacologically relevant pre-clinical doses. The findings highlight metformin's rapid brain entry, minimal binding, and metabolic stability. The necessity of considering pharmacokinetic parameters in pre-clinical studies provides a foundation for future investigations into metformin's efficacy for neurodegenerative disease (s).

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.

Discovery of the Next Generation of Non-peptidomimetic Neurolysin Activators with High Blood-Brain Barrier Permeability: a Pharmacokinetics Study in Healthy and Stroke Animals.

PURPOSE: There is growing interest in seeking pharmacological activation of neurolysin (Nln) for stroke treatment. Discovery of central nervous system drugs remains challenging due to the protection of the blood-brain barrier (BBB). The previously reported peptidomimetic Nln activators display unsatisfactory BBB penetration. Herein, we investigate the next generation of non-peptidomimetic Nln activators with high BBB permeability. METHODS: A BBB-mimicking model was used to evaluate their in vitro BBB permeability. Protein binding, metabolic stability, and efflux assays were performed to determine their unbound fraction, half-lives in plasma and brains, and dependence of BBB transporter P-glycoprotein (P-gp). The in vivo pharmacokinetic profiles were elucidated in healthy and stroke mice. RESULTS: Compounds KS52 and KS73 out of this generation exhibit improved peptidase activity and BBB permeability compared to the endogenous activator and previous peptidomimetic activators. They show reasonable plasma and brain protein binding, improved metabolic stability, and independence of P-gp-mediated efflux. In healthy animals, they rapidly distribute into brains and reach peak levels of 18.69% and 12.10% injected dose (ID)/ml at 10 min. After 4 h, their total brain concentrations remain 7.78 and 12.34 times higher than their A50(minimal concentration required for enhancing 50% peptidase activity). Moreover, the ipsilateral hemispheres of stroke animals show comparable uptake to the corresponding contralateral hemispheres and healthy brains. CONCLUSIONS: This study provides essential details about the pharmacokinetic properties of a new generation of potent non-peptidomimetic Nln activators with high BBB permeability and warrants the future development of these agents as potential neuroprotective pharmaceutics for stroke treatment.

Impact of in-utero electronic cigarette exposure on neonatal neuroinflammation, oxidative stress and mitochondrial function.

Introduction: Despite the prevalence of the perception that electronic cigarettes (e-cig) are a safer alternative to tobacco smoke, growing concern about their potential toxic impact warrants adequate investigation focusing on special populations like maternal and pediatric groups. This study evaluated the consequences of maternal e-cig use on neonatal neuroinflammation, oxidative stress, and mitochondrial function in primary cultured neurons and postnatal day (PD) 7 and 90 brain. Methodology: Pregnant CD1 mice were exposed to e-cig vapor (2.4% nicotine) from gestational day 5 (E5) till PD7, and the primary neurons were isolated from pups at E16/17. Cellular total reactive oxygen species (ROS) and mitochondrial superoxide were measured in primary neurons using CM-H2DCFDA and Mitosox red, respectively. Mitochondrial function was assessed by Seahorse XF Cell Mitostress analysis. The level of pro-inflammatory cytokines was measured in primary neurons and PD7 and PD90 brains by RT-PCR and immunobead assay. Western blot analysis evaluated the expression of antioxidative markers (SOD-2, HO-1, NRF2, NQO1) and that of the proinflammatory modulator NF-κB. Results: Significantly higher level of total cellular ROS (p < 0.05) and mitochondrial superoxide (p < 0.01) was observed in prenatally e-cig-exposed primary neurons. We also observed significantly reduced antioxidative marker expression and increased proinflammatory modulator and cytokines expression in primary neurons and PD7 (p < 0.05) but not in PD90 postnatal brain. Conclusion: Our findings suggest that prenatal e-cig exposure induces postnatal neuroinflammation by promoting oxidative stress (OS), increasing cytokines' levels, and disrupting mitochondrial function. These damaging events can alter the fetal brain's immune functions, making such offspring more vulnerable to brain insults.

Studies on diketopiperazine and dipeptide analogs as opioid receptor ligands.

Using the structure of gliotoxin as a starting point, we have prepared two different chemotypes with selective affinity to the kappa opioid receptor (KOR). Using medicinal chemistry approaches and structure-activity relationship (SAR) studies, structural features required for the observed affinity were identified, and advanced molecules with favorable Multiparameter Optimization (MPO) and Ligand Lipophilicity (LLE) profiles were prepared. Using the Thermal Place Preference Test (TPPT), we have shown that compound2 blocks the antinociceptive effect of U50488, a known KOR agonist. Multiple reports suggest that modulation of KOR signaling is a promising therapeutic strategy in treating neuropathic pain (NP). As a proof-of-concept study, we tested compound 2 in a rat model of NP and recorded its ability to modulate sensory and emotional pain-related behaviors. Observed in vitro and in vivo results suggest that these ligands can be used to develop compounds with potential application as pain therapeutics.

Maternal e-cigarette use can disrupt postnatal blood-brain barrier (BBB) integrity and deteriorates motor, learning and memory function: influence of sex and age.

Electronic nicotine delivery systems (ENDS), also commonly known as electronic cigarettes (e-cigs) are considered in most cases as a safer alternative to tobacco smoking and therefore have become extremely popular among all age groups and sex. It is estimated that up to 15% of pregnant women are now using e-cigs in the US which keeps increasing at an alarming rate. Harmful effects of tobacco smoking during pregnancy are well documented for both pregnancy and postnatal health, however limited preclinical and clinical studies exist to evaluate the long-term effects of prenatal e-cig exposure on postnatal health. Therefore, the aim of our study is to evaluate the effect of maternal e-cig use on postnatal blood-brain barrier (BBB) integrity and behavioral outcomes of mice of varying age and sex. In this study, pregnant CD1 mice (E5) were exposed to e-Cig vapor (2.4% nicotine) until postnatal day (PD) 7. Weight of the offspring was measured at PD0, PD7, PD15, PD30, PD45, PD60 and PD90. The expression of structural elements of the BBB, tight junction proteins (ZO-1, claudin-5, occludin), astrocytes (GFAP), pericytes (PDGFRß) and the basement membrane (laminin α1, laminin α4), neuron specific marker (NeuN), water channel protein (AQP4) and glucose transporter (GLUT1) were analyzed in both male and female offspring using western blot and immunofluorescence. Estrous cycle was recorded by vaginal cytology method. Long-term motor and cognitive functions were evaluated using open field test (OFT), novel object recognition test (NORT) and morris water maze test (MWMT) at adolescence (PD 40-45) and adult (PD 90-95) age. In our study, significantly reduced expression of tight junction proteins and astrocyte marker were observed in male and female offspring until PD 90 (P < 0.05). Additionally, prenatally e-cig exposed adolescent and adult offspring showed impaired locomotor, learning, and memory function compared to control offspring (P < 0.05). Our findings suggest that prenatal e-cig exposure induces long-term neurovascular changes of neonates by disrupting postnatal BBB integrity and worsening behavioral outcomes.

Endothelial-Specific Targeting of RhoA Signaling via CD31 Antibody-Conjugated Nanoparticles.

Existing vascular endothelial growth factor-oriented antiangiogenic approaches are known for their high potency. However, significant side effects associated with their use drive the need for novel antiangiogenic strategies. The small GTPase RhoA is an established regulator of actin cytoskeletal dynamics. Previous studies have highlighted the impact of endothelial RhoA pathway on angiogenesis. Rho-associate kinase (ROCK), a direct RhoA effector, is potently inhibited by Fasudil, a clinically relevant ROCK inhibitor. Here, we aimed to target the RhoA signaling in endothelial cells by generating Fasudil-encapsulated CD31-targeting liposomes as a potential antiangiogenic therapy. The liposomes presented desirable characteristics, preferential binding to CD31-expressing HEK293T cells and to endothelial cells, inhibited stress fiber formation and cytoskeletal-related morphometric parameters, and inhibited in vitro angiogenic functions. Overall, this work shows that the nanodelivery-mediated endothelial targeting of RhoA signaling can offer a promising strategy for angiogenesis inhibition in vascular-related diseases. SIGNIFICANCE STATEMENT: Systemic administration of antiangiogenic therapeutics induces side effects to non-targeted tissues. This study, among others, has shown the impact of the RhoA signaling in the endothelial cells and their angiogenic functions. Here, to minimize potential toxicity, this study generated CD31-targeting liposomes with encapsulated Fasudil, a clinically relevant Rho kinase inhibitor, and successfully targeted endothelial cells. In this proof-of-principle study, the efficient Fasudil delivery, its impact on the endothelial signaling, morphometric alterations, and angiogenic functions verify the benefits of site-targeted antiangiogenic therapy.

In Vivo Evaluation of BBB Integrity in the Post-stroke Brain.

The blood-brain barrier (BBB) is a dynamic interface responsible for maintaining central nervous system (CNS) homeostasis. An intact BBB protects the brain from undesired compounds and proteins from the blood; however, BBB impairment is involved in various pathological conditions including stroke. In vivo evaluation of BBB integrity in the post-stroke brain is important for investigating stroke-induced CNS pathogenesis and developing CNS-targeted therapeutic agents. In this chapter, we describe both quantitative and morphometric methods and tools to evaluate BBB integrity in vivo. These methods do not require expensive magnetic resonance imaging (MRI) and computed tomography (CT) imaging capabilities and can be conducted in research laboratories with access to a confocal microscope and fluorescence microplate reader.

Dental Pulp-Derived Stem Cells Preserve Astrocyte Health During Induced Gliosis by Modulating Mitochondrial Activity and Functions.

Astrocytes have been implicated in the onset and complication of various central nervous system (CNS) injuries and disorders. Uncontrolled astrogliosis (gliosis), while a necessary process for recovery after CNS trauma, also causes impairments in CNS performance and functions. The ability to preserve astrocyte health and better regulate the gliosis process could play a major role in controlling damage in the aftermath of acute insults and during chronic dysfunction. Here in, we demonstrate the ability of dental pulp-derived stem cells (DPSCs) in protecting the health of astrocytes during induced gliosis. First of all, we have characterized the expression of genes in primary astrocytes that are relevant to the pathological conditions of CNS by inducing gliosis. Subsequently, we found that astrocytes co-cultured with DPSCs reduced ROS production, NRF2 and GCLM expressions, mitochondrial membrane potential, and mitochondrial functions compared to the astrocytes that were not co-cultured with DPSCs in gliosis condition. In addition, hyperactive autophagy was also decreased in astrocytes that were co-cultured with DPSCs compared to the astrocytes that were not co-cultured with DPSCs during gliosis. This reversal and mitigation of gliosis in astrocytes were partly due to induction of neurogenesis in DPSCs through enhanced expressions of the neuronal genes like GFAP, NeuN, and Synapsin in DPSCs and by secretion of higher amounts of neurotropic factors, such as BDNF, GDNF, and TIMP-2. Protein-Protein docking analysis suggested that BDNF and GDNF can bind with CSPG4 and block the downstream signaling. Together these findings demonstrate novel functions of DPSCs to preserve astrocyte health during gliosis.

Short-term exposure to JUUL electronic cigarettes can worsen ischemic stroke outcome.

BACKGROUND: The short and long-term health effects of JUUL electronic cigarette (e-Cig) are largely unknown and warrant extensive research. We hypothesized that JUUL exposure could cause cerebrovascular toxicities impacting the progression and outcome of ischemic stroke comparable to tobacco smoke (TS) exposure. METHODS: We exposed male C57 mice to TS/JUUL vapor for 14 days. LCMS/MS was used to measure brain and plasma nicotine and cotinine level. Transient middle cerebral artery occlusion (tMCAO) followed by reperfusion was used to mimic ischemic stroke. Plasma levels of IL-6 and thrombomodulin were assessed by enzyme-linked immunosorbent assay. At the same time, western blotting was used to study blood-brain barrier (BBB) tight junction (TJ) proteins expression and key inflammatory and oxidative stress markers. RESULTS: tMCAO upregulated IL-6 and decreased plasma thrombomodulin levels. Post-ischemic brain injury following tMCAO was significantly worsened by JUUL/TS pre-exposure. TJ proteins expression was also downregulated by JUUL/TS pre-exposure after tMCAO. Like TS, exposure to JUUL downregulated the expression of the antioxidant Nrf2. ICAM-1 was upregulated in mice subjected to tMCAO following pre-exposure to TS or JUUL, with a greater effect of TS than JUUL. CONCLUSIONS: These results suggest that JUUL exposure could negatively impact the cerebrovascular system, although to a lesser extent than TS exposure.

Brain Energy Metabolism in Ischemic Stroke: Effects of Smoking and Diabetes.

Proper regulation of energy metabolism in the brain is crucial for maintaining brain activity in physiological and different pathophysiological conditions. Ischemic stroke has a complex pathophysiology which includes perturbations in the brain energy metabolism processes which can contribute to worsening of brain injury and stroke outcome. Smoking and diabetes are common risk factors and comorbid conditions for ischemic stroke which have also been associated with disruptions in brain energy metabolism. Simultaneous presence of these conditions may further alter energy metabolism in the brain leading to a poor clinical prognosis after an ischemic stroke event. In this review, we discuss the possible effects of smoking and/or diabetes on brain glucose utilization and mitochondrial energy metabolism which, when present concurrently, may exacerbate energy metabolism in the ischemic brain. More research is needed to investigate brain glucose utilization and mitochondrial oxidative metabolism in ischemic stroke in the presence of smoking and/or diabetes, which would provide further insights on the pathophysiology of these comorbid conditions and facilitate the development of therapeutic interventions.

Potential role of astrocyte angiotensin converting enzyme 2 in the neural transmission of COVID-19 and a neuroinflammatory state induced by smoking and vaping.

BACKGROUND: Knowledge of the entry receptors responsible for SARS-CoV-2 is key to understand the neural transmission and pathogenesis of COVID-19 characterized by a neuroinflammatory scenario. Understanding the brain distribution of angiotensin converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, remains mixed. Smoking has been shown as a risk factor for COVID-19 severity and it is not clear how smoking exacerbates the neural pathogenesis in smokers. METHODS: Immunohistochemistry, real-time PCR and western blot assays were used to systemically examine the spatial-, cell type- and isoform-specific expression of ACE2 in mouse brain and primary cultured brain cells. Experimental smoking exposure was conducted to evaluate the effect of smoking on brain expression. RESULTS: We observed ubiquitous expression of ACE2 but uneven brain distribution, with high expression in the cerebral microvasculature, medulla oblongata, hypothalamus, subventricular zones, and meninges around medulla oblongata and hypothalamus. Co-staining with cell type-specific markers demonstrates ACE2 is primarily expressed in astrocytes around the microvasculature, medulla oblongata, hypothalamus, ventricular and subventricular zones of cerebral ventricles, and subependymal zones in rhinoceles and rostral migratory streams, radial glial cells in the lateral ventricular zones, tanycytes in the third ventricle, epithelial cells and stroma in the cerebral choroid plexus, as well as cerebral pericytes, but rarely detected in neurons and cerebral endothelial cells. ACE2 expression in astrocytes is further confirmed in primary cultured cells. Furthermore, isoform-specific analysis shows astrocyte ACE2 has the peptidase domain responsible for SARS-CoV-2 entry, indicating astrocytes are indeed vulnerable to SARS-CoV-2 infection. Finally, our data show experimental tobacco smoking and electronic nicotine vaping exposure increase proinflammatory and/or immunomodulatory cytokine IL-1a, IL-6 and IL-5 without significantly affecting ACE2 expression in the brain, suggesting smoking may pre-condition a neuroinflammatory state in the brain. CONCLUSIONS: The present study demonstrates a spatial- and cell type-specific expression of ACE2 in the brain, which might help to understand the acute and lasting post-infection neuropsychological manifestations in COVID-19 patients. Our data highlights a potential role of astrocyte ACE2 in the neural transmission and pathogenesis of COVID-19. This also suggests a pre-conditioned neuroinflammatory and immunocompromised scenario might attribute to exacerbated COVID-19 severity in the smokers.

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.

Glutamate Buffering Capacity and Blood-Brain Barrier Protection of Opioid Receptor Agonists Biphalin and Nociceptin.

Opioids play crucial roles in the regulation of many important brain functions including pain, memory, and neurogenesis. Activation of opioid receptors is reported to have neuroprotective effects after ischemic reperfusion injury. The objective of this study was to understand the role of biphalin and nociceptin, opioid receptor agonists, on blood-brain barrier (BBB) integrity during ischemic stroke. In this study, we aimed to measure the effect of biphalin and nociceptin on astrocytic glutamate uptake and on expression of excitatory amino acid transporter to study the indirect role of astrocytes on opioid receptor-mediated BBB protection during in vitro stroke conditions. We used mouse brain endothelial cells (bEnd.3) and primary astrocytes as an in vitro BBB model. Restrictive BBB properties were evaluated by measuring [14C] sucrose paracellular permeability and the redistribution of the tight junction proteins. The protective effect of biphalin and nociceptin on BBB integrity was assessed after exposing cells to oxygen glucose deprivation (OGD) and glutamate. It was observed that combined stress (2 mM glutamate and 2 hours of OGD) significantly reduced glutamate uptake by astrocytes; however, biphalin and nociceptin treatment increased glutamate uptake in primary astrocytes. This suggests a role of increased astrocytic buffering capacity in opioid-meditated protection of the BBB during ischemic stroke. It was also found that the combined stress significantly increased [14C] sucrose paracellular permeability in an in vitro BBB model. Biphalin and nociceptin treatment attenuated the effect of the combined stress, which was reversed by the opioid receptor antagonists, suggesting the role of opioid receptors in biphalin and nociception's BBB modulatory activity. SIGNIFICANT STATEMENT: There is an unmet need for discovering new efficacious therapeutic agents to offset the deleterious effects of ischemic stroke. Given the confirmed roles of opioid receptors in the regulation of central nervous system functions, opioid receptor agonists have been studied as potential neuroprotective options in ischemic conditions. This study adds to the knowledge about the cerebrovascular protective effects of opioid receptor agonists and provides insight about the mechanism of action of these agents.

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.

Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of Neurolysin.

Neurolysin (Nln) is a recently recognized endogenous mechanism functioning to preserve the brain from ischemic injury. To further understand the pathophysiological function of this peptidase in stroke and other neurologic disorders, the present study was designed to identify small molecule activators of Nln. Using a computational approach, the structure of Nln was explored, which was followed by docking and in silico screening of ∼140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database. Top ranking compounds were evaluated in an Nln enzymatic assay, and two hit histidine-dipeptides were further studied in detail. The identified dipeptides enhanced the rate of synthetic substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Nln in a concentration-dependent manner (micromolar A50 and Amax ≥ 300%) but had negligible effect on activity of closely related peptidases. Both dipeptides also enhanced hydrolysis of Nln endogenous substrates neurotensin, angiotensin I, and bradykinin and increased efficiency of the synthetic substrate hydrolysis (Vmax/Km ratio) in a concentration-dependent manner. The dipeptides and competitive inhibitor dynorphin A (1-13) did not affect each other's affinity for Nln, suggesting differing nature of their respective binding sites. Lastly, drug affinity responsive target stability (DARTS) and differential scanning fluorimetry (DSF) assays confirmed concentration-dependent interaction of Nln with the activator molecule. This is the first study demonstrating that Nln activity can be enhanced by small molecules, although the peptidic nature and low potency of the activators limit their application. The identified dipeptides provide a chemical scaffold to develop high-potency, drug-like molecules as research tools and potential drug leads. SIGNIFICANCE STATEMENT: This study describes discovery of two molecules that selectively enhance activity of peptidase Nln-a newly recognized cerebroprotective mechanism in the poststroke brain. The identified molecules will serve as a chemical scaffold for development of drug-like molecules to further study Nln and may become lead structures for a new class of drugs. In addition, our conceptual and methodological framework and research findings might be used for other peptidases and enzymes, the activation of which bears therapeutic potential.