Design and synthesis of novel kinesin spindle protein

The design of synthetic small molecules that target cytoskeleton microtubule is important for the investigation of the mechanism of cell division and for the development of novel anti-tumor agents.;Molecules targeting microtubule stability such as Taxanes and Vinca alkaloids are among the most successful anticancer agents. However, such molecules cause undesired side effects, including neurotoxicity and are subject to acquired drug resistance. In 1999, researchers discovered a completely new target for anti-mitotic activity is the microtubule-associated kinesin Eg5 (kinesin spindle protein, KSP). Eg5 is a member of the kinesin-5 family and plays an important role in the early stages of mitosis. Specifically, the kinesin Eg5 plays an important role in bipolar spindle formation. Inhibition of this motor protein leads to the formation of monopolar spindles, mitotic arrest, followed by cell apoptosis. Molecules targeting KSP selectively act only on the cells undergoing cell division, making them mitosis-specific drugs. Therefore, KSP has become a novel and attractive target for cancer treatment.;S-trityl-L-cysteine (STLC) is a potent allosteric inhibitor of kinesin spindle protein (KSP); however, this compound has limited potential use for cancer treatment due to amphiphilic and poor drug-like characteristics. We have modified the triphenylmethyl and cysteine groups in STLC and synthesized new cysteinol and cysteamine derivatives guided by biochemical, cell-based assays, and have utilized molecular docking techniques to understand the structure-activity relationships of these novel inhibitors. Several of our compounds show increased potency in both biochemical and cell-based assays. Two of our active compounds were evaluated for anti-proliferative activity against the NCI60 tumor panel and showed a 143- and 35-fold average increase in potency over Monastrol and STLC, respectively. In addition, these analogs had activities over a broad range of tumor types, representing a significant increase in anti-proliferative potency.;Our group has synthesized a new class of fluorescent triaza-borolopyridinium compounds from hydrazones of 2-hydrazinylpyridine (HPY). These dyes are small, have easily tunable absorption/ emission properties through variation of pyridyl or hydrazone substituents. The stability, neutral charge, cell membrane permeability and relative water solubility of HPY conjugates offer advantages for the development of receptor-targeted small-molecule probes. To study the live cell imaging applications of HPY dyes, we synthesized a new class of KSP targeting imaging probes. This novel synthetic dye conjugates demonstrated potent activity against KSP.;To understand the possible binding modes of novel triphenylmethyl derivatives, we performed molecular docking experiments using the crystal structure of human KSP (PDB Id: 2FME) by using computational modeling software tools. Active molecules were docked into the binding pocket of KSP with the ammonium group forming strong hydrogen-bonding interactions with Glu 116 and Gly 117, and the three aromatic rings of the trityl group were oriented in the allosteric hydrophobic pockets. Ligand structures were generated using the software Spartan'10 and docking studies were performed using the software programs Gold Suite 5.0.1 and Hermes 1.4.1. The highest scoring poses were visualized by using software Ligand Scout 3.0.;Considering alternative structures related to triphenylmethyl, we became interested in cationic triphenyl phosphonium (TPP) analogs as potential KSP inhibitors. These TPP analogs are isosteres of the trityl group containing three-aryl substituents and a primary amine. We predicted that this new class of TPP analogs might be able to inhibit KSP, and should have increased water solubility and membrane permeability due to the presence of the overall positive charge on phosphorous. Docking experiments also predicted favorable KSP binding of TPP analogs, with relatively large GOLD scores. However, TPP analogs were inactive in the biochemical ATPase assay and in the cell-based assay. Further docking studies demonstrated that the three-phenyl rings of TPP analogs are able to bind in the same pocket, but they could not elicit an allosteric response due to their inability to form a key interaction with tryptophan residue 127 that is required to close the loop L5.;This work demonstrates the rational design, synthesis and structure-activity relationship studies of novel triphenylmethyl derivatives as KSP inhibitors. The potent activity and conformational flexibility of these small allosteric inhibitors of KSP offers real promise as research tools and as lead compounds for the development of clinically useful anti-tumor agents.