Discovery of novel inhibitors of histone deacetylase 6: Structure-based virtual screening, molecular dynamics simulation, enzyme inhibition and cell viability assays.

Histone deacetylase 6 (HDAC6) contributes to cancer metastasis in several cancers, including triple-negative breast cancer (TNBC)-the most lethal form that lacks effective therapy. Although several efforts have been invested to develop selective HDAC6 inhibitors, none have been approved by the FDA. Toward this goal, existing computational studies used smaller compound libraries and shorter MD simulations. Here, we conducted a structure-based virtual screening of ZINC "Druglike" library containing 17,900,742 compounds using a Glide virtual screening protocol comprising various filters with increasing accuracy. The top 20 hits were subjected to molecular dynamics simulation, MM-GBSA binding energy calculations, and further ADMET prediction. Furthermore, enzyme inhibition assay and cell viability assay were performed on six available compounds from the identified hits. C4 (ZINC000077541942) with a good profile of predicted drug properties was found to inhibit HDAC6 (IC50: 4.7 ± 11.6 µM) with comparative affinity to that of the known HDAC6 selective inhibitor Tubacin (TA) in our experiments. C4 also demonstrated cytotoxic effects against triple-negative breast cancer (TNBC) cell line MDA-MB-231 with EC50 of 40.6 ± 12.7 µM comparable to that of TA (2-20 µM). Therefore, this compound, with pharmacophore features comprising a non-hydroxamic acid zinc-binding group, heteroaromatic linker, and cap group, is proposed as a novel HDAC6 inhibitor.

L-shaped distribution of the relative substitution rate (c/µ) observed for SARS-COV-2's genome, inconsistent with the selectionist theory, the neutral theory and the nearly neutral theory but a near-neutral balanced selection theory: Implication on "neutralist-selectionist" debate.

The genomic substitution rate (GSR) of SARS-CoV-2 exhibits a molecular clock feature and does not change under fluctuating environmental factors such as the infected human population (10°-107), vaccination etc. The molecular clock feature is believed to be inconsistent with the selectionist theory (ST). The GSR shows lack of dependence on the effective population size, suggesting Ohta's nearly neutral theory (ONNT) is not applicable to this virus. Big variation of the substitution rate within its genome is also inconsistent with Kimura's neutral theory (KNT). Thus, all three existing evolution theories fail to explain the evolutionary nature of this virus. In this paper, we proposed a Segment Substitution Rate Model (SSRM) under non-neutral selections and pointed out that a balanced mechanism between negative and positive selection of some segments that could also lead to the molecular clock feature. We named this hybrid mechanism as near-neutral balanced selection theory (NNBST) and examined if it was followed by SARS-CoV-2 using the three independent sets of SARS-CoV-2 genomes selected by the Nextstrain team. Intriguingly, the relative substitution rate of this virus exhibited an L-shaped probability distribution consisting with NNBST rather than Poisson distribution predicted by KNT or an asymmetric distribution predicted by ONNT in which nearly neutral sites are believed to be slightly deleterious only, or the distribution that is lack of nearly neutral sites predicted by ST. The time-dependence of the substitution rates for some segments and their correlation with the vaccination were observed, supporting NNBST. Our relative substitution rate method provides a tool to resolve the long standing "neutralist-selectionist" controversy. Implications of NNBST in resolving Lewontin's Paradox is also discussed.

Binding of a positive allosteric modulator CDPPB to metabotropic glutamate receptor type 5 (mGluR5) probed by all-atom molecular dynamics simulations.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor type 5 (mGluR5) potentiate positive receptor response and may be effective for the treatment of schizophrenia and cognitive disorders. Although crystal structures of mGluR5 complexed with the negative allosteric modulators (NAMs) are available, no crystal structure of mGluR5 complexed with PAM has been reported to date. Thus, conformational changes associated with the binding of PAMs to mGluR5 remain elusive. Here, a PAM CDPPB, and two NAMs MTEP and MFZ10-7 used as a negative control, were docked to the crystal structure. The docked complexes were submitted to molecular dynamics simulations to examine the activation of the PAM system. An MM/GBSA binding energy calculation was performed to estimate binding strength. Furthermore, molecular switch analysis was done to get insights into conformational changes of the receptor. The PAM CDPPB displays a stronger binding affinity for mGluR5 and induces conformational changes. Also, a salt bridge between TM3 and TM7, corresponding to the ionic lock switch in class A GPCRs is found to be broken. The PAM-induced receptor conformation is more like the agonist-induced conformation than the antagonist-induced conformation, suggesting that PAM works by inducing conformation change and stabilizing the active receptor conformation.