Discovery of tetrahydro-β-carbolines as inhibitors of the mitotic kinesin KSP

Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest in cells from a failure to form functional bipolar mitotic spindles. Here, we report the synthesis and biological evaluation of a novel series of tetrahydro-β-carboline analogs based on the structure of the known KSP inhibitor HR22C16. Preferred compounds 11b, 12a and 19b were identified as potent inhibitors in a KSP ATPase assay with good anti-proliferative activity in A549 cells.     

Kinesin spindle protein (KSP) inhibitors. Part 1: The discovery of 3,5-diaryl-4,5-dihydropyrazoles as potent and selective inhibitors of the mitotic kinesin KSP

Optimization of high-throughput screening (HTS) hits resulted in the discovery of 3,5-diaryl-4,5-dihydropyrazoles as potent and selective inhibitors of KSP. Dihydropyrazole 15 is a potent, cell-active KSP inhibitor that induces apoptosis and generates aberrant mitotic spindles in human ovarian carcinoma cells at low nanomolar concentrations. X-ray crystallographic evidence is presented which demonstrates that these inhibitors bind in an allosteric pocket of KSP distant from the nucleotide and microtubule binding sites.     

Synthesis and anticancer evaluation of 3-substituted quinolin-4-ones and 2,3-dihydroquinolin-4-ones

A series of 3-aryl-5,7-dimethoxyquinolin-4-ones 8 and 3-aryl-5,7-dimethoxy-2,3-dihydroquinolin-4-ones 13 were synthesized in good yields. Demethylation under a range of conditions afforded the corresponding 5-hydroxy and 5,7-dihydroxy derivatives. Biological evaluation against a range of cancer cells lines showed that the quinolin-4-one scaffold was more cytotoxic than the reduced 2,3-dihydroquinolin-4-one scaffold. The most active monohydroxy compound 15f demonstrated 85.9–99% reduction in cell viability against the cell lines tested.     

Synthesis and biological evaluation of new tetrahydro-beta-carbolines as inhibitors of the mitotic kinesin Eg5

The mitotic kinesin Eg5 (or KSP) is a crucial player in the development and function of the mitotic spindle. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering with other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, we report the synthesis and biological evaluation of a small library of molecules based on the structure of the known Eg5 inhibitor HR22C16. One of these derivatives (compound trans-24) proved to be a potent and specific Eg5 inhibitor.     

The Synthesis and Evaluation of Dihydroquinazolin-4-ones and Quinazolin-4-ones as Thyroid Stimulating Hormone Receptor Agonists

We herein describe the rapid synthesis of a diverse set of dihydroquinazolin-4-ones and quinazolin-4-ones, their biological evaluation as thyroid stimulating hormone receptor (TSHR) agonists, and SAR analysis. Among the compounds screened, 8b was 60-fold more potent than the hit compound 1a, which was identified from a high throughput screen of over 73,000 compounds.     

Bis(hetero)aryl derivatives as unique kinesin spindle protein inhibitors

Synthesis of 4-(4-tert-butylphenyl)pyridine analogues as kinesin spindle protein (KSP) inhibitors, SAR, cytotoxicity and mitotic arrest in HeLa cells are described. Interestingly, PVZB1194 showed potent KSP inhibition only in the presence of microtubules and distinct KSP localization from a known KSP inhibitor S-trytylcysteine analogue in mitosis. The observations would have resulted from a different molecular mechanism of KSP inhibition and suggest a novel biological regulation for KSP in mitosis.     

Mechanism of inhibition of human KSP by ispinesib

KSP, also known as HsEg5, is a kinesin that plays an essential role in the formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. Ispinesib is the first potent, highly specific small-molecule inhibitor of KSP tested for the treatment of human disease. This novel anticancer agent causes mitotic arrest and growth inhibition in several human tumor cell lines and is currently being tested in multiple phase II clinical trials. In this study we have used steady-state and pre-steady-state kinetic assays to define the mechanism of KSP inhibition by ispinesib. Our data show that ispinesib alters the ability of KSP to bind to microtubules and inhibits its movement by preventing the release of ADP without preventing the release of the KSP-ADP complex from the microtubule. This type of inhibition is consistent with the physiological effect of ispinesib on cells, which is to prevent KSP-driven mitotic spindle pole separation. A comparison of ispinesib to monastrol, another small-molecule inhibitor of KSP, reveals that both inhibitors share a common mode of inhibition.     

Discovery and biochemical characterization of selective ATP competitive inhibitors of the human mitotic kinesin KSP

The kinesin spindle protein (KSP, also known as Eg5) is essential for the proper separation of spindle poles during mitosis, and inhibition results in mitotic arrest and the formation of characteristic monoaster spindles. Several distinct classes of KSP inhibitors have been described previously in the public and patent literature. However, most appear to share a common induced-fit allosteric binding site, suggesting a common mechanism of inhibition. In a high-throughput screen for inhibitors of KSP, a novel class of thiazole-containing inhibitors was identified. Unlike the previously described allosteric KSP inhibitors, the thiazoles described here show ATP competitive kinetic behavior, consistent with binding within the nucleotide binding pocket. Although they bind to a pocket that is highly conserved across kinesins, these molecules exhibit significant selectivity for KSP over other kinesins and other ATP-utilizing enzymes. Several of these compounds are active in cells and produce a phenotype similar to that observed with previously published allosteric inhibitors of KSP.     

Synthesis and structure-activity relationships of benzophenone-bearing diketopiperazine-type anti-microtubule agents

KPU-105 (4), a potent anti-microtubule agent that contains a benzophenone was derived from the diketopiperazine-type vascular disrupting agent (VDA) plinabulin 3, which displays colchicine-like tubulin depolymerization activity. To develop derivatives with more potent anti-microtubule and cytotoxic activities, we further modified the benzophenone moiety of 4. Accordingly, we obtained a 4-fluorobenzophenone derivative 16j that inhibited tumor cell growth in vitro with a subnanomolar IC(50) value against HT-29 cells (IC(50)=0.5 nM). Next, the effect of 16j on mitotic spindles was evaluated in HeLa cells. Treatment with 3nM of 16j partially disrupted the interphase microtubule network. By contrast, treatment with the same concentration of CA-4 barely affected the microtubule network, indicating that 16j exhibited more potent anti-mitotic effects than did CA-4.     

Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.     

BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo

Fine-mapping of the cell-division cycle, notably the identification of mitotic kinase signaling pathways, provides novel opportunities for cancer-drug discovery. As a key regulator of multiple steps during mitotic progression across eukaryotic species, the serine/threonine-specific Polo-like kinase 1 (Plk1) is highly expressed in malignant cells and serves as a negative prognostic marker in specific human cancer types . Here, we report the discovery of a potent small-molecule inhibitor of mammalian Plk1, BI 2536, which inhibits Plk1 enzyme activity at low nanomolar concentrations. The compound potently causes a mitotic arrest and induces apoptosis in human cancer cell lines of diverse tissue origin and oncogenome signature. BI 2536 inhibits growth of human tumor xenografts in nude mice and induces regression of large tumors with well-tolerated intravenous dose regimens. In treated tumors, cells arrest in prometaphase, accumulate phosphohistone H3, and contain aberrant mitotic spindles. This mitotic arrest is followed by a surge in apoptosis, detectable by immunohistochemistry and noninvasive optical and magnetic resonance imaging. For addressing the therapeutic potential of Plk1 inhibition, BI 2536 has progressed into clinical studies in patients with locally advanced or metastatic cancers.     

Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity

Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer properties, however, poor pharmacological properties have limited the use of this compound. We have modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a promising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as research tools, with activities that provide impetus for further development as anti-tumor agents.     

Astrin regulates Aurora-A localization

Alterations in the expression and activity of the centrosomal kinase, Aurora-A/STK15, affect genomic stability, disrupt the fidelity of centrosome duplication, and induce cellular transformation. A mitotic spindle-associated protein, astrin/DEEPEST, was identified as an Aurora-A interacting protein by a two-hybrid screen. Astrin and Aurora-A co-express at mitosis and co-localize to mitotic spindles. RNAi-mediated depletion of astrin abolishes the localization of Aurora-A on mitotic spindles and leads to a moderate mitotic cell cycle delay, which resembles the mitotic arrest phenotypes in siAurora-A treated cells. However, depletion of Aurora-A does not affect astrin localization, and co-depletion of both astrin and Aurora-A causes a mitotic arrest phenotype similar to depletion of siAurora-A alone. These results suggest that astrin acts upstream of Aurora-A to regulate its mitotic spindle localization.     

Synthesis and Evaluation of Biphenyl Compounds as Kinesin Spindle Protein Inhibitors

Kinesin spindle protein (KSP), an ATP‐dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression. Antiproliferation (MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide)) assays, combined with fluorescence‐assisted cell sorting (FACS) and Western blot studies analyzing cell‐cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Structural variants revealed that functionalization of biphenyl compounds with bulky aliphatic or aromatic groups led to a loss of activity. However, replacement of the urea group with a thiourea led to an increase in antiproliferative activity in selected cell lines. Further studies using confocal fluorescence microscopy confirmed that the most potent biphenyl derivative identified thus far, compound 7 , exerts its pharmacologic effect specifically in the M phase and induces monoaster formation. These studies confirm that chemical scope remains for improving the potency and treatment efficacy of antimitotic KSP inhibition in this class of biphenyl compounds.     

Synthesis and biological evaluation of L-cysteine derivatives as mitotic kinesin Eg5 inhibitors

Inhibition of Eg5 represents a novel approach for the treatment of cancer. Here, we report the synthesis and structure-activity relationship of S-trityl-L-cysteine (STLC) derivatives as Eg5 inhibitors. Some of these derivatives such as 4f demonstrated enhanced inhibitory activity against Eg5 and induced mitotic arrest with characteristic monoastral spindles in HeLa cells.     

3,5-Dihydro-imidazo[4,5-d]pyridazin-4-ones: a class of potent DPP-4 inhibitors

Systematic variations of the xanthine scaffold in close analogs of development compound BI 1356 led to the class of 3,5-dihydro-imidazo[4,5-d]pyridazin-4-ones which provided, after substituent screening, a series of highly potent DPP-4 inhibitors.     

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug''s actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers), and vice versa (suppressors), and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens, and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology.     

3-Alkyl- and 3-amido-isothiazoloquinolin-4-ones as ligands for the benzodiazepine site of GABA(A) receptors

Based on a pharmacophore model of the benzodiazepine binding site of the GABA(A) receptors, developed with synthetic flavones and potent 3-carbonylquinolin-4-ones, 3-alkyl- and 3-amido-6-methylisothiazoloquinolin-4-ones were designed, prepared and assayed. The suggestion that the interaction between the hydrogen bond donor site H1 with the 3-carbonyl oxygen in 3-carbonylquinolin-4-ones can be replaced by an interaction between H1 and N-2 in the isothiazoloquinolin-4-ones, was confirmed. As with the 3-carbonylquinolin-4-ones, the length of the chain in position 3 is critical for an efficient interaction with the lipophilic pockets of the pharmacophore model. The most potent 3-alkyl derivative, 3-pentyl-6-methylisothiazoloquinolin-4-one, has an affinity (K(i) value) for the benzodiazepine binding site of the GABA(A) receptors of 13 nM. However, by replacing the 3-pentyl with a 3-butyramido group an even more potent compound was obtained, with a K(i) value of 2.8 nM, indicating that the amide function facilitates additional interactions with the binding site.     

Mechanism of mitotic arrest induced by dolastatin 15 involves loss of tension across kinetochore pairs

Dolastatin 15 (DL15) is a potent, tubulin-targeted, vinca-site binding, anticancer agent that induces mitotic arrest and inhibit cell proliferation in a variety of cell types. Several analogs of DL15, including LU 103793 and tasidotin, have been progressed to clinical trials for different types of cancer. DL15 has been known to interfere with cellular microtubules and purified tubulin in vitro. However, the molecular mechanism with which the peptide arrests cells in mitosis is poorly understood. This study reports a possible antimitotic mechanism of action of DL15. DL15 inhibited HeLa cell proliferation in a concentration-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 2.8 ± 0.3 nM, induced mitotic arrest, disrupted cellular microtubules near its IC₅₀ for cell proliferation, and inhibited the re-polymerization of cellular microtubules. By staining the centrosomes of DL15-treated cells with anti-γ tubulin antibodies, the study found a significant reduction in interpolar distances in mitotic HeLa cells, indicating a disruption in the normal assembly dynamics of the microtubules. The study further found that DL15 induced a loss of tension across the kinetochore pairs as indicated by a reduction in interkinetochore distance. In response to this loss of tension, the tension-sensing checkpoint protein BuBR1 accumulated at the kinetochores, promoting mitotic arrest. In vitro, DL15 promoted formation of curved and fragmented polymers of microtubule proteins and inhibited tubulin decay in a manner similar to vinca-site binding agents such as phomopsin A. Together, the data indicate that the mitotic arrest induced by DL15 involves a loss of tension across the kinetochore pairs due to disruption of normal assembly dynamics of microtubules.