Empirical Analysis of Drug Targets for Nervous System Disorders

The discovery and development of drugs to treat diseases of the nervous system remains challenging. There is a higher attrition rate in the clinical stage for nervous system experimental drugs compared to other disease areas. In the preclinical stage, additional challenges arise from the considerable effort required to find molecules that penetrate the blood–brain barrier (BBB) coupled with the poor predictive value of many preclinical models of nervous system diseases. In the era of target-based drug discovery, the critical first step of drug discovery projects is the selection of a therapeutic target which is largely driven by its presumed pathogenic involvement. For nervous system diseases, however, the feasibility of identifying potent molecules within the stringent range of molecular properties necessary for BBB penetration should represent another important factor in target selection. To address the latter, the present review analyzes the distribution of human protein targets of FDA-approved drugs for nervous system disorders and compares it with drugs for other disease areas. We observed a substantial difference in the distribution of therapeutic targets across the two clusters. We expanded on this finding by analyzing the physicochemical properties of nervous and non-nervous system drugs in each target class by using the central nervous system multiparameter optimization (CNS MPO) algorithm. These data may serve as useful guidance in making more informed decisions when selecting therapeutic targets for nervous system disorders

GCP II inhibition improves hot plate reaction times.

<p>Pyridoxine intoxication increased reaction time on the hot plate, which was improved by GCP II on days 18–25. By day 29, both of the pyridoxine treated groups had recovered to control levels. N = 10/group. Data = mean ± SEM. * = p<0.05.</p

Timeline of experiment.

<p>Timeline of experiment.</p

GCP II inhibition did not significantly affect weight loss induced by pyridoxine.

<p>Weights were measured every day of the study. A significant difference was observed between the controls and both the pyridoxine treated groups (p<0.05); no difference was observed between the pyridoxine/vehicle and pyridoxine/2-MPPA groups (p>0.05). N = 10/group. Data = mean ± SEM. * = p<0.05.</p

Effects of 6‑Aminonicotinic Acid Esters on the Reprogrammed Epigenetic State of Distant Metastatic Pancreatic Carcinoma

In the search for alternatives to 6-aminonicotinamide (6AN), a series of 6-aminonicotinic acid esters were designed and synthesized as precursors of 6-amino-NADP+, a potent inhibitor of 6-phosphogluconate dehydrogenase (6PGD). Like 6AN, some of these esters were found to reverse the loss of histone 3 lysine 9 trimethylation (H3K9me3) in patient-derived pancreatic ductal adenocarcinoma (PDAC) distant metastasis (A38-5). Among them, 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 6-aminonicotinate (5i) showed more potent antiproliferative activity than 6AN. Metabolite analysis revealed that compound 5i produced a marked increase in metabolites upstream of 6PGD, indicating intracellular inhibition of 6PGD by 6-amino-NADP+ derived from compound 5i through 6-aminonicotinic acid (6ANA) via the Preiss–Handler pathway. Despite the more potent pharmacological effects shown by compound 5i in A38-5, compound 5i was found to be substantially less toxic to primary hippocampal rat neurons compared to 6AN, indicating its therapeutic potential in targeting distant metastatic cells

Initial screening results of the compounds in the LOPAC and NINDS chemical libraries using the cystine-induced glutamate release assay.

<p>CCF-STTG-1 cells were seeded and grown to confluence. On Day 3, cells were washed with pre-warmed EBSS and transport initiated upon the addition of cystine (80 μM). Cystine-induced glutamate released over 2h at 37°C, in the presence and absence of inhibitors, was measured directly using glutamate oxidase, horse radish peroxidase and Amplex UltraRed and the rate of change of fluorescence monitored at ex 530, em 590. Results were normalized to totals and blanks and the IC₅₀ values determined as a function of the normalized values.</p

Selective CNS Uptake of the GCP-II Inhibitor 2-PMPA following Intranasal Administration

<div><p>Glutamate carboxypeptidase II (GCP-II) is a brain metallopeptidase that hydrolyzes the abundant neuropeptide N-acetyl-aspartyl-glutamate (NAAG) to NAA and glutamate. Small molecule GCP-II inhibitors increase brain NAAG, which activates mGluR3, decreases glutamate, and provide therapeutic utility in a variety of preclinical models of neurodegenerative diseases wherein excess glutamate is presumed pathogenic. Unfortunately no GCP-II inhibitor has advanced clinically, largely due to their highly polar nature resulting in insufficient oral bioavailability and limited brain penetration. Herein we report a non-invasive route for delivery of GCP-II inhibitors to the brain via intranasal (i.n.) administration. Three structurally distinct classes of GCP-II inhibitors were evaluated including DCMC (urea-based), 2-MPPA (thiol-based) and 2-PMPA (phosphonate-based). While all showed some brain penetration following i.n. administration, 2-PMPA exhibited the highest levels and was chosen for further evaluation. Compared to intraperitoneal (i.p.) administration, equivalent doses of i.n. administered 2-PMPA resulted in similar plasma exposures (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 1.0) but dramatically enhanced brain exposures in the olfactory bulb (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 67), cortex (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 46) and cerebellum (AUC_{0-t, i.n}./AUC_{0-t, i.p.} = 6.3). Following i.n. administration, the brain tissue to plasma ratio based on AUC_0-t in the olfactory bulb, cortex, and cerebellum were 1.49, 0.71 and 0.10, respectively, compared to an i.p. brain tissue to plasma ratio of less than 0.02 in all areas. Furthermore, i.n. administration of 2-PMPA resulted in complete inhibition of brain GCP-II enzymatic activity <i>ex-vivo</i> confirming target engagement. Lastly, because the rodent nasal system is not similar to humans, we evaluated i.n. 2-PMPA also in a non-human primate. We report that i.n. 2-PMPA provides selective brain delivery with micromolar concentrations. These studies support intranasal delivery of 2-PMPA to deliver therapeutic concentrations in the brain and may facilitate its clinical development.</p></div

Dose responses of A. sulfasalazine (SAS), B. (<i>S</i>)-4-carboxyphenylglycine ((<i>S</i>)-4CPG) and C. (<i>R</i>)-4-carboxyphenylglycine ((<i>R</i>)-4CPG) using [¹⁴C]-cystine uptake and cystine-induced glutamate release assays in CCF-STTG-1 cells.

<p>Cystine uptake was conducted at 37°C for 15 min using 80μM cystine, and at a specific activity of 5.631 μCi/μmol, in the presence and absence of inhibitors. At the end of the experiment, cells were lysed and the radioactivity in the cells measured using a scintillation counter and normalized to the protein contents. Cystine-induced glutamate released over 2h at 37°C upon the addition of 80 μM cystine, in the presence and absence of inhibitors, was measured directly using glutamate oxidase, horse radish peroxidase and Amplex UltraRed and the rate of change of fluorescence monitored at ex 530, em 590. For both assays, results were normalized to totals and blanks and the IC₅₀ values determined as a function of the normalized values. Data are an average of 2–6 independent experiments.</p

Chemical structures and IC₅₀ values of DCMC, 2-MPPA, 2-PMPA.

<p>Chemical structures and IC₅₀ values of DCMC, 2-MPPA, 2-PMPA.</p

Dependence of cystine-induced glutamate release in CCF-STTG-1 cells at 37°C on A. cystine concentration, B. cystine concentration represented via Lineweaver-Burk transformation C. protein concentration (mg/ml) and D. time of incubation.

<p>Unless otherwise specified, CCF-STTG-1 cells were seeded at 0.04 x 10⁶ cells per 96-well, grown to confluence (Day 3), washed with pre-warmed EBSS and transport initiated upon the addition of cystine. Substrate dependence experiments were carried out with 0–400 μM cystine while protein- and time-dependence experiments were carried out with 80 μM cystine. Cells were maintained for 2h at 37°C in an incubator with 5% CO₂. At the end of the incubation period, glutamate release was measured directly using glutamate oxidase (0.04 U/mL), HRP (0.125 U/mL) and Amplex UltraRed (50 μM), in Tris buffer (100 mM, pH 7.4), at ex 530, em 590. Data are an average of 3 independent experiments with 16–24 determinations per experiment.</p