Novel photochromic inhibitor for mitotic kinesin Eg5 which forms multiple isomerization states

The mitotic kinesin Eg5 is a plus-end directed homotetrameric molecular motor essential for the formation of bipolar spindles during cell division. Kinesin Eg5 is overexpressed in cancer cells and hence considered as a target for cancer therapy; the inhibitors specific for Eg5 have been developed as anticancer drugs. In this study, we synthesized a novel functional photoresponsive inhibitor composed of spiropyran and azobenzene derivatives to control Eg5 function with multistage inhibitory activity accompanied by the formation of different isomerization states. The photochromic inhibitor spiropyran-sulfo-azobenzene (SPSAB) exhibited three isomerization states: spiro (SP)-trans, merocyanine (MC)-cis and MC-trans, upon exposure to visible light, ultraviolet and in the dark, respectively. SPSAB-induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with photoisomerization among the three states. Among the three isomerization states of SPSAB, the SP-trans isomer showed potent inhibitory activity at an IC50 value of 30 µM in the basal ATPase assay. MC-trans and MC-cis exhibited less inhibitory activity at IC50 values of 38 and 86 µM, respectively. The results demonstrated that the novel photochromic inhibitor enabled precise control of Eg5 function at three different levels using light irradiation.

Evaluating Phenyl Propanoids Isolated from Citrus medica as Potential Inhibitors for Mitotic kinesin Eg5

Background: Human mitotic kinesins play an essential role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention in cancer chemotherapy. Spindle segregation is carried out mainly by the kinesin, Eg5. Many Eg5 inhibitors are in different phases of clinical trials as cancer drugs. This enzyme has two allosteric binding sites to which the inhibitors can bind. The first site is formed by loop L5, helix α2 and helix α3 and all the current drug candidates bind un-competitively to this site with ATP/ADP. The second site, formed by helix α4 and helix α6, which has gained attention recently, has not been explored well. Some inhibitors that bind to this site are competitive, while others are uncompetitive to ATP/ADP. Phenylpropanoids are pharmacologically active secondary metabolites. Methods: In this study, we have evaluated fourteen phenyl propanoids extracted from Citrus medica for inhibitory activity against human mitotic kinesin Eg5 in vitro steady-state ATPase assay. Ther interactions and stability using molecular docking and molecular dynamics simulations. Results and Discussions: Of the fourteen compounds tested, naringin and quercetin showed good activity with IC50 values in the micromolar range. Molecular docking studies of these complexes showed that both the molecules interact with the key residues of the active site predominantly thorough hydrophobic & aromatic π–π interactions consistent with the known inhibitors. Besides, these molecules also form hydrogen bonding interactions stabilizing the complexes. Molecular dynamics simulations of these complexes confirm the stability of these interactions. Conclusion: These results can be used as a strong basis for further modification of these compounds to design new inhibitors with higher potency using structure-based drug design.

Lead Generation for Human Mitotic Kinesin Eg5 using Structure-based Virtual Screening and Validation by in vitro and Cell-based Assays

Background: Human mitotic kinesins play a crucial role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention pharmaceutically in cancer chemotherapy. Spindle segregation is carried out mainly by Eg5 kinesin, and currently, it has many inhibitors in different phases of clinical trials. All the current drug candidates bind un-competitively with ATP/ADP at allosteric site 1 (formed by loop L5, helix α2 and helix α3). Recent experiments show that inhibitors that bind to the site 2 (formed by helix α4 and helix α6) are either competitive or uncompetitive to ATP/ADP. Objectives: To identify suitable lead compounds that target the mitotic kinesin Eg5, using in silico screening and their validation using in vitro and cell-based assays. Methodology: We have screened for potential inhibitors for human Eg5 (kinesin-5) through structure-based virtual screening and validated the top-scoring compounds using steady-state ATPase assay, differential scanning fluorimetry and microscale thermophoresis. The anticancer activity of the compounds was evaluated in the epithelial (A549) and chronic myelogenous leukaemia (K562) cancer cell lines. A known strong binding inhibitor S-trityl-L-cystine is used as a reference compound. Results & Conclusion: Of the many compounds tested, MM01 and MM03 showed good cell-based activity against the cancer cell lines A549 and K562 and can be further studied in animal models.

Antiproliferative S-Trityl-l-Cysteine -Derived Compounds as SIRT2 Inhibitors: Repurposing and Solubility Enhancement

S-trityl-l-cysteine (STLC) is a well-recognized lead compound known for its anticancer activity owing to its potent inhibitory effect on human mitotic kinesin Eg5. STLC contains two free terminal amino and carboxyl groups that play pivotal roles in binding to the Eg5 pocket. On the other hand, such a zwitterion structure complicates the clinical development of STLC because of the solubility issues. Masking either of these radicals reduces or abolishes STLC activity against Eg5. We recently identified and characterized a new class of nicotinamide adenine dinucleotide-dependent deacetylase isoform 2 of sirtuin protein (SIRT2) inhibitors that can be utilized as cytotoxic agents based on an S-trityl-l-histidine scaffold. Herein, we propose new STLC-derived compounds that possess pronounced SIRT2 inhibition effects. These derivatives contain modified amino and carboxyl groups, which conferred STLC with SIRT2 bioactivity, representing an explicit repurposing approach. Compounds STC4 and STC11 exhibited half maximal inhibitory concentration values of 10.8 ± 1.9 and 9.5 ± 1.2 μM, respectively, against SIRT2. Additionally, introduction of the derivatizations in this study addressed the solubility limitations of free STLC, presumably due to interruption of the zwitterion structure. Therefore, we could obtain drug-like STLC derivatives that work by a new mechanism of action. The new derivatives were designed, synthesized, and their structure was confirmed using different spectroscopic approaches. In vitro and cellular bioassays with various cancer cell lines and in silico molecular docking and solubility calculations of the synthesized compounds demonstrated that they warrant attention for further refinement of their bioactivity.

Morelloflavone as a novel inhibitor of mitotic kinesin Eg5

Among 40 plant-derived biflavonoids with inhibitory potential against Eg5, morelloflavone from Garcinia dulcis leaves was selected for further testing based on in silico analysis of binding modes, molecular interactions, binding energies and functional groups that interact with Eg5. Computational models predicted that morelloflavone binds the putative allosteric pocket of Eg5, within the cavity surrounded by amino acid residues of Ile-136, Glu-116, Glu-118, Trp-127, Gly-117, Ala-133, Glu-215, Leu-214 and Tyr-211. Binding energy was −8.4 kcal/mol, with a single hydrogen bond formed between morelloflavone and Tyr-211. The binding configuration was comparable to that of a reference inhibitor, S-trityl-L-cysteine. Subsequent biochemical analysis in vitro confirmed that morelloflavone inhibited both the basal and microtubule-activated ATPase activity of Eg5 in a manner that does not compete with ATP binding. Morelloflavone also suppressed Eg5 gliding along microtubules. These results suggest that morelloflavone binds the allosteric binding site in Eg5 and thereby inhibits ATPase activity and motor function of Eg5.