Virtual screening identification of novel chemical inhibitors for aberrant interactions between pathogenic mutant SOD1 and tubulin.

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease caused by selective motor neuron death. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) belong to one of the four major mutation clusters responsible for pathogenesis of ALS. Toxic gain-of-function (not loss-of-function) of SOD1 mutants causes motor neuron degeneration. Aberrant protein-protein interactions (PPI) between mutant SOD1 and other proteins are involved in this toxic gain-of-function. Therefore, PPI inhibitors of mutant SOD1 not only increase understanding of ALS pathogenesis, but can also be used as novel therapeutics for ALS. Although it is challenging to identify PPI inhibitors, prior knowledge of the binding site can increase success probability. We have previously reported that tubulin interacts with N-terminal residues 1-23 of mutant SOD1. In the present study, we performed virtual screening by docking simulation of 32,791 compounds using this N-terminal binding site as prior knowledge. An established assay system for interaction inhibition between mutant SOD1-tubulin was used as an in-house model system to identify mutant SOD1 PPI inhibitors, with the goal of developing novel therapeutics for ALS. Consequently, five of six assay-executable compounds among top-ranked compounds during docking simulation inhibited the mutant SOD1-tubulin interaction in vitro. Binding mode analysis predicted that some inhibitors might bind the tubulin binding site of G85R SOD1 by pi electron interaction with the aromatic ring of the Trp32 residue of G85R SOD1. Our screening methods may contribute to the identification of lead compounds for treating ALS.

Antibody informatics for drug discovery.

More and more antibody therapeutics are being approved every year, mainly due to their high efficacy and antigen selectivity. However, it is still difficult to identify the antigen, and thereby the function, of an antibody if no other information is available. There are obstacles inherent to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii) antibody numbering and IMGT. Here, we review "antibody informatics," which may integrate the above three fields so that bridging the gaps between industrial needs and academic solutions can be accelerated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

The L-, N-, and T-type triple calcium channel blocker benidipine acts as an antagonist of mineralocorticoid receptor, a member of nuclear receptor family.

Aldosterone-induced activation of mineralocorticoid receptor, a member of the nuclear receptor family, results in increased tissue damage such as vascular inflammation and cardiac and perivascular fibrosis. Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for hypertension and angina. Benidipine exhibits pleiotropic pharmacological features such as renoprotective and cardioprotective effects through triple blockade of L-, N-, and T-type calcium channels. However, the mechanism of additional beneficial effects on end-organ damage is poorly understood. Here, we examined the effects of benidipine and other calcium channel blockers on aldosterone-induced mineralocorticoid receptor activation using luciferase reporter assay system. Benidipine showed more potent activity than efonidipine, amlodipine, or azelnidipine. Benidipine depressed the response to higher concentrations of aldosterone, whereas pretreatment of eplerenone, a steroidal mineralocorticoid receptor antagonist, did not. Binding studies using [(3)H] aldosterone indicated that benidipine and other calcium channel blockers competed for binding to mineralocorticoid receptor. Benidipine and other calcium channel blockers showed antagonistic activity on Ser810 to Leu mutant mineralocorticoid receptor, which is identified in patients with early-onset hypertension. On the other hand, eplerenone partially activated the mutant. Results of analysis using optical isomers of benidipine indicated that inhibitory effect of aldosterone-induced mineralocorticoid receptor activation was independent of its primary blockade of calcium channels. These results suggested that benidipine directly inhibits aldosterone-induced mineralocorticoid receptor activation, and the antagonistic activity might contribute to the drug's pleiotropic pharmacological features.

Identification of a novel chemical potentiator and inhibitors of UCH-L1 by in silico drug screening.

Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a de-ubiquitinating enzyme expressed in the brain and reproductive tissues as well as certain cancers. The hydrolase activity of UCH-L1 has been implicated in Alzheimer's disease and cancer invasion; therefore, it may represent a therapeutic target for these diseases. The present study was undertaken to identify novel chemical modulators for the hydrolase activity of UCH-L1. To identify chemicals that bind to the active site of UCH-L1, we carried out in silico structure-based drug screening using human UCH-L1 crystal structure data (PDB ID: 2ETL) and virtual compound libraries containing 26,891 and 304,205 compounds. Among the compounds with the highest binding scores, we identified one that potentiates the hydrolase activity of UCH-L1, and six that inhibit the activity in enzymatic assays. These compounds may be useful for research on UCH-L1 function, and could lead to candidate therapeutics for UCH-L1-associated diseases.

Identification of novel chemical inhibitors for ubiquitin C-terminal hydrolase-L3 by virtual screening.

UCH-L3 (ubiquitin C-terminal hydrolase-L3) is a de-ubiquitinating enzyme that is a component of the ubiquitin-proteasome system and known to be involved in programmed cell death. A previous study of high-throughput drug screening identified an isatin derivative as a UCH-L3 inhibitor. In this study, we attempted to identify a novel inhibitor with a different structural basis. We performed in silico structure-based drug design (SBDD) using human UCH-L3 crystal structure data (PDB code; 1XD3) and the virtual compound library (ChemBridge CNS-Set), which includes 32,799 chemicals. By a two-step virtual screening method using DOCK software (first screening) and GOLD software (second screening), we identified 10 compounds with GOLD scores of over 60. To address whether these compounds exhibit an inhibitory effect on the de-ubiquitinating activity of UCH-L3, we performed an enzymatic assay using ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) as the substrate. As a result, we identified three compounds with similar basic dihydro-pyrrole skeletons as UCH-L3 inhibitors. These novel compounds may be useful for the research of UCH-L3 function, and in drug development for UCH-L3-associated diseases.