Development of First-in-Class Dual Sirt2/HDAC6 Inhibitors as Molecular Tools for Dual Inhibition of Tubulin Deacetylation.

Dysregulation of both tubulin deacetylases sirtuin 2 (Sirt2) and the histone deacetylase 6 (HDAC6) has been associated with the pathogenesis of cancer and neurodegeneration, thus making these two enzymes promising targets for pharmaceutical intervention. Herein, we report the design, synthesis, and biological characterization of the first-in-class dual Sirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulin deacetylation. Using biochemical in vitro assays and cell-based methods for target engagement, we identified Mz325 (33) as a potent and selective inhibitor of both target enzymes. Inhibition of both targets was further confirmed by X-ray crystal structures of Sirt2 and HDAC6 in complex with building blocks of 33. In ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to single or combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus, our dual Sirt2/HDAC6 inhibitors are important new tools to study the consequences and the therapeutic potential of dual inhibition of tubulin deacetylation.

Design, synthesis and biological evaluation of novel hybrids targeting mTOR and HDACs for potential treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major contributor to global cancer incidence and mortality. Many pathways are involved in the development of HCC and various proteins including mTOR and HDACs have been identified as potential drug targets for HCC treatment. In the present study, two series of novel hybrid molecules targeting mTOR and HDACs were designed and synthesized based on parent inhibitors (MLN0128 and PP121 for mTOR, SAHA for HDACs) by using a fusion-type molecular hybridization strategy. In vitro antiproliferative assays demonstrated that these novel hybrids with suitable linker lengths exhibited broad cytotoxicity against various cancer cell lines, with significant activity against HepG2 cells. Notably, DI06, an MLN0128-based hybrid, exhibited antiproliferative activity against HepG2 cells with an IC50 value of 1.61 µM, which was comparable to those of both parent drugs (MLN0128, IC50 = 2.13 µM and SAHA, IC50 = 2.26 µM). In vitro enzyme inhibition assays indicated that DI06, DI07 and DI17 (PP121-based hybrid) exhibited nanomolar inhibitory activity against mTOR kinase and HDACs (e.g., HDAC1, HDAC2, HDAC3, HADC6 and HADC8). Cellular studies and western blot analyses uncovered that in HepG2 cells, DI06 and DI17 induced cell apoptosis by targeting mTOR and HDACs, blocked the cell cycle at the G0/G1 phase and suppressed cell migration. The potential binding modes of the hybrids (DI06 and DI17) with mTOR and HDACs were investigated by molecular docking. DI06 displayed better stability in rat liver microsomes than DI07 and DI17. Collectively, DI06 as a novel mTOR and HDACs inhibitor presented here warrants further investigation as a potential treatment of HCC.

Ligand- and structural-based discovery of potential small molecules that target the colchicine site of tubulin for cancer treatment.

Small molecules targeting the colchicine site of tubulin represent an attractive cancer treatment strategy. In this study, a total of 468 models derived from 1076 diverse inhibitors binding to the tubulin colchicine site were constructed based on fingerprints using three machine learning approaches: 1) naive Bayesian (NB); 2) single tree (ST); and 3) random forest (RF). The overall predictive accuracy of the best models exceeded 85.12% for both the training and test sets. We designed an integrated virtual screening (VS) strategy for identifying new tubulin inhibitors by combining established models, molecular docking, and similarity-based analog searching. Through two rounds of VS, compound 23g was identified as a novel potent anticancer agent exhibiting activity against MDA-MB-231, HeLa, A549, HepG2, CNE2, and HCT116 tumor cell lines with IC50 values of 5.45, 8.61, 7.47, 2.29, 2.91, and 4.10 µM, respectively. Compared with taxol, colchicine, and adriamycin, 23g also displayed potent cytotoxicity against the drug-resistant tumor cell lines, HepG2/ADR, A549/CDDP, and A549/TAX cells, with IC50 values of 4.12, 6.58, and 6.38 µM, respectively. Further mechanistic studies revealed that 23g inhibited microtubule polymerization by binding to the colchicine site of tubulin, arrested the cell cycle at the G2/M phase, induced cell apoptosis, and exhibited potent in vitro anti-metastasis activity. Finally, molecular docking, molecular dynamics, and free energy analyses were employed to explore the detailed binding interaction between 23g and tubulin. Collectively, these findings indicated that 23g should be further investigated as a potential novel potent antitumor agent targeting tubulin.

HybridMolDB: A Manually Curated Database Dedicated to Hybrid Molecules for Chemical Biology and Drug Discovery.

Hybrid-molecule-based drug design is the combination of two or more bioactive molecules into a single chemical entity. This strategy may be used to achieve better affinity and efficacy or improved properties compared with the parent molecules, to interact with two or multiple targets, to reduce undesirable side effects, to decrease drug-drug interactions, or to reduce the emergence of drug resistance. The approach offers the prospect of better drugs for the treatment of many human diseases. Research activity in this area is increasing and has attracted many practitioners worldwide. To accelerate the design and discovery of new hybrid-molecule-based drugs, it is essential to properly collect and annotate experimental data obtained from known hybrid molecules. To address this need, we have developed HybridMolDB ( http://www.idruglab.com/HybridMolDB/index.php ), a manually curated database dedicated to hybrid molecules for chemical biology and drug discovery. It contains structures, manually annotated design protocols, pharmacological data, some physicochemical properties, ligand efficiency, drug-likeness, and ADMET characteristics, and the biological targets of known hybrid molecules. HybridMolDB supports a range of query types, including searches by text, protein sequence, chemical structure similarity, and property ranges. The database serves as an open source facilitating the development and/or optimization of related in silico tools for the design and discovery of hybrid-molecule-based drugs and chemical probes.

Discovery, biological evaluation, structure-activity relationships and mechanism of action of pyrazolo[3,4-b]pyridin-6-one derivatives as a new class of anticancer agents.

We have recently reported computational models for prediction of cell-based anticancer activity using machine learning methods. Herein, we have developed an integrated strategy to discover new anticancer agents using a cascade of the established screening models. Application of this strategy identified 17 compounds with antitumor activity. Among these compounds, h2 (containing a pyrazolo[3,4-b]pyridin-6-one scaffold) exhibited anticancer activity against six tumor cell lines, including MDA-MB-231, HeLa, MCF-7, HepG2, CNE2 and HCT116, with IC50 values of 13.37, 13.04, 15.45, 7.05, 9.30 and 8.93 µM. Subsequently, a total of 61 h2 analogues were obtained by similarity searching and tested for their anticancer activities. I2 was identified as a novel anticancer agent having activity against MDA-MB-231, HeLa, MCF-7, HepG2, CNE2 and HCT116 tumor cell lines with IC50 values of 3.30, 5.04, 5.08, 3.71, 2.99 and 5.72 µM. I2 also showed potent cytotoxicity against adriamycin-resistant human breast and hepatocarcinoma cells. Further investigation revealed that I2 inhibited the microtubule polymerization by binding to the colchicine site, resulting in inhibition of cell migration, cell cycle arrest in the G2/M phase and apoptosis of cancer cells. Finally, molecular docking and molecular dynamics provided insights into the binding interactions of I2 with tubulin. This study identified I2 as a novel starting point for further development of anticancer agents that target tubulin.

Structure-activity relationship and synthetic methodologies of α-santonin derivatives with diverse bioactivities: A mini-review.

α-Santonin, a sesquiterpene lactone isolated from Artemisia Santonica, possesses diverse bioactivities including antioxidant, anti-inflammation, immunosuppressive, anti-roundworm, anti-malaria, etc. However, its bioactivities are not satisfactory and need to be further optimized. Thus, many α-santonin derivatives were synthesized on the basis of rings A, B and C for the discovery of new analogues with prominent bioactivities. Herein, we reviewed and discussed the related synthetic methodologies, diverse bioactivities and structure-activity relationships (SAR) of α-santonin derivatives.