βIII Isotype Research Articles

PM534, an Optimized Target-Protein Interaction Strategy through the Colchicine Site of Tubulin.

Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure-Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the order Lithistida, family Theonellidae, genus Discodermia (du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model. Its nanomolar affinity and high retention time modulate a strikingly high antitumor activity that efficiently overrides two resistance mechanisms in cells (detoxification pumps and tubulin βIII isotype overexpression). Furthermore, PM534 induces significant inhibition of tumor growth in mouse xenograft models of human non-small cell lung cancer. Our results present PM534, a highly effective new compound in the preclinical evaluation that is currently in its first human Phase I clinical trial.

Possible Roles of Specific Amino Acids in β-Tubulin Isotypes in the Growth and Maintenance of Neurons: Novel Insights From Cephalopod Mollusks.

Microtubules, are formed of the protein tubulin, which is a heterodimer of α- and β-tubulin subunits. Both α- and β-tubulin exist as numerous isotypes, differing in amino acid sequence and tissue distribution. Among the vertebrate β isotypes, βIII has a very narrow distribution, being found primarily in neurons and in advanced cancers. The places in the amino acid sequence where βIII differs from the other β isotypes are highly conserved in evolution. βIII appears to be highly resistant to reactive oxygen species and it forms highly dynamic microtubules. The first property would be very useful in neurons, which have high concentrations of free radicals, and the high dynamicity would aid neurite outgrowth. The same properties make βIII useful in cancers. Examination of the amino acid sequences indicates a cysteine cluster at positions 124–129 in βIII (CXXCXC). This occurs in all βIII isotypes but not in βI, βII, or βIV. βIII also lacks the easily oxidized C239. Both features could play roles in free radical resistance. Many aggressive tumors over-express βIII. However, a recent study of breast cancer patients showed that many of them mutated their βI, βII, and βIV at particular places to change the residues to those found at the corresponding sites in βIII; these are all sites that are highly conserved in vertebrate βIII. It is possible that these residues are important, not only in the resistance to free radicals, but also in the high dynamicity of βIII. The cephalopod mollusks are well known to be highly intelligent and can remodel their own brains. Interestingly, several cephalopods contain the cysteine cluster as well as up to 7 of the 17 residues that are highly conserved in vertebrate βIII, but are not found in βI, βII, or βIV. In short, it is possible that we are looking at a case of convergent evolution, that a βIII-like isotype may be required for neuronal growth and function and that a structure-function study of the particular residues conserved between vertebrate βIII and cephalopod tubulin isotypes could greatly increase our understanding of the role of the various tubulin isotypes in neuronal growth and function and could aid in the development of novel anti-tumor drugs.

Exploring the interaction of Peloruside-A with drug resistant αβII and αβIII tubulin isotypes in human ovarian carcinoma using a molecular modeling approach

Microtubules (MTs) play an essential role in mitosis; hence they are identified as potential targets to design novel anti-mitotic agents. MT’s are composed of α/β-tubulin isotypes that are associated with differential drug-resistant effects against MT-targeting agents. Peloruside-A (PLA) is a potent anti-mitotic agent showing excellent activity against taxol-resistant carcinoma. PLA alters MT dynamics by binding to the ‘non-taxoid’ site of β-tubulin. The abundance of βII and βIII tubulin isotypes in human ovarian carcinoma affects the efficacy of PLA. Nevertheless, the mechanism of PLA resistance due to βII and βIII tubulin isotype is not well understood. Therefore, we investigated the interactions of PLA with αβIIa, αβIIb, and αβIII tubulin isotypes which are predominantly expressed in the human ovarian carcinoma, using a molecular modeling approach. A sequence analysis study shows that the βIII isotype has seven residue variations at the ‘non-taxoid’ site compared to the βIIa and βIIb isotypes. Molecular docking and molecular dynamics simulation revealed that residue variation at the ‘non-taxoid’ site of βIII isotype affect PLA binding. Furthermore, binding energy calculations showed that αβIIa has the highest binding towards PLA, whereas αβIIb and αβIII isotypes shows weaker associations with PLA. Our computational study provides valuable structural and energetic information to increase understanding into the origin of PLA resistance in human ovarian carcinoma and could be helpful to develop potential PLA analogs against specific β-tubulin isotypes expressed in cancer cells. Communicated by Ramaswamy H. Sarma

Unravelling the covalent binding of zampanolide and taccalonolide AJ to a minimalist representation of a human microtubule.

Many natural products target mammalian tubulin but only a few can form a covalent bond and hence irreversibly affect microtubule function. Among them, zampanolide (ZMP) and taccalonolide AJ (TAJ) stand out, not only because they are very potent antitumor agents but also because the adducts they form with β-tubulin have been structurally characterized in atomic detail. By applying model building techniques, molecular orbital calculations, molecular dynamics simulations and hybrid QM/MM methods, we have gained insight into the 1,2- and 1,4-addition reactions of His229 and Asp226 to ZMP and TAJ, respectively, in the taxane-binding site of β-tubulin. The experimentally inaccessible precovalent complexes strongly suggest a water-mediated proton shuttle mechanism for ZMP adduct formation and a direct nucleophilic attack by the carboxylate of Asp226 on C22 of the C22R,C23R epoxide in TAJ. The M-loop, which is crucially important for interprotofilament interactions, is structured into a short helix in both types of complexes, mostly as a consequence of the fixation of the phenol ring of Tyr283 and the guanidinium of Arg284. As a side benefit, we obtained evidence supporting the existence of a commonly neglected intramolecular disulfide bond between Cys241 and Cys356 in β-tubulin that contributes to protein compactness and is absent in the βIII isotype associated with resistance to taxanes and other drugs.

Delineating the interaction of combretastatin A-4 with αβ tubulin isotypes present in drug resistant human lung carcinoma using a molecular modeling approach

Tubulin isotypes are known to regulate microtubule dynamic instability and contribute to the development of drug resistance in certain types of cancers. Combretastatin-A4 (CA-4) has a potent anti-mitotic, vascular disrupting and anti-angiogenic activity. It binds at the interface of αβ tubulin heterodimers and inhibits microtubules assembly. Interestingly, the CA-4 resistant human lung carcinoma shows alteration of βI and βIII isotype levels, a higher expression of βI tubulin isotype and a decreased expression of βIII tubulin isotypes has been reported in drug resistant cell lines. However, the origin of CA-4 resistance in lung carcinoma is not well understood. Here, we investigate the interaction and binding affinities of αβI, αβIIb, αβIII and αβIVa tubulin isotypes with CA-4, employing molecular modeling approaches. Sequence analysis shows that variations in residue composition at the CA-4 binding pocket of βI, βIII and βIVa tubulin isotypes when compared to template βIIb isotype. Molecular docking result shows that the CA-4 prefers ‘cis’ conformation in all αβ-tubulin isotypes. Molecular dynamics simulation reveal role of H7 helix, T7 loop and H8 helix of β-tubulin in lower binding affinity of αβI and αβIII isotypes for CA-4. The order of binding energy for CA-4 is αβIIb > αβIVa > αβI > αβIII. This suggest that drug resistance is induced in human lung carcinoma cells by altering the expression of β-tubulin isotypes namely βI and βIII which show lowest binding affinities. Our present study can help in designing potential CA-4 analogs against drug-resistant cancer cells showing altered expression of tubulin isotypes.Abbreviations:CA-4combretastatin-A4MDmolecular dynamicsRMSDroot mean square deviationDSSPdictionary of secondary structure of proteinsVMDvisual molecular dynamicsCommunicated by Ramaswamy H. Sarma

Abstract 4851: Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors

Abstract We have developed a class of small-molecule cytotoxic compounds that are highly potent in an in vitro tubulin polymerization assay. Preclinical studies show that these drug candidates are potent against a range of cancer cell lines and may be well suited for the treatment of hematologic or solid tumors resistant to existing chemotherapy agents. We will present data on representative members of this class that inhibit cancer cell growth at nanomolar concentrations and are potent against a variety of cancer cell lines, including liver, breast, ovarian, and lung. The compounds induce cell cycle arrest in the G2M state within 24 hours with cell death occurring over multiple days. These tubulin inhibitors also behave as potent antiangiogenesis agents and inhibit endothelial tube formation in HUVEC cells at nanomolar concentrations. Functional assays using efflux pump inhibitors show that, compared to many major chemotherapy agents, our class of drug candidates is significantly less affected by the efflux pumps MDR1 and MRP1, which are commonly overexpressed as a mechanism of multidrug resistance. Our compounds show comparable potency against wild-type and cell lines developed to be resistant to other cancer drugs, while the potency of clinically relevant compounds such as paclitaxel or doxorubicin is reduced between 80x and about 3000x in the same assay. This feature suggests that our tubulin inhibitors may be active against tumors that are resistant to common cancer drugs. Overexpression of the β-III isotype of tubulin, which is known to occur in many aggressive and metastatic tumors, is another clinically relevant mechanism of resistance to microtubule-targeting anticancer agents. It has been correlated with significantly lower response to docetaxel-based chemotherapy in a number of cancers and is considered an indicator of resistance to paclitaxel and vinorelbine. We will present preclinical data showing that our compounds have similar activity against cells showing normal levels and those highly overexpressing β-III tubulin. The compounds display good in vitro physicochemical properties as well as favorable in vivo pharmacokinetics. In addition, we will present in vivo tolerability and efficacy data in mice. The ability of this class of tubulin inhibitors to maintain their efficacy across multiple drug-resistant cancer cell lines makes them attractive candidates for development as chemotherapy agents. In particular, a new anticancer agent that is less susceptible to major transporters and retains its potency when β-III tubulin is overexpressed could lead to more effective precision second-line therapy. Citation Format: Mohan Sivaraja, Sivan Sizikov, Nilantha Sirisoma, Tamari Kirtadze, Madhuri Chattopadhyay, Makena Ewald, Subhadra Dash, Anne Wong, Georg Neckermann, Elaine To, Stephanie Chang, Timothy P. Shiau, David C. Williams, Kevin M. Short, Angels Estiarte, Anirban Datta, David B. Kita. Potent cell cycle inhibitors suitable for treatment of multidrug-resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4851.

Interactions of β tubulin isotypes with glutathione in differentiated neuroblastoma cells subject to oxidative stress.

Microtubules are a major component of the neuronal cytoskeleton. Tubulin, the subunit protein of microtubules, is an α/β heterodimer. Both α and β exist as families of isotypes, whose members are encoded by different genes and have different amino acid sequences. The βII and βIII isotypes are very prominent in the nervous system. Our previous work has suggested that βII may play a role in neuronal differentiation, but the role of βIII in neurons is not well understood. In the work reported here, we examined the roles of the different β-tubulin isotypes in response to glutamate/glycine treatment, and found that both βII and βIII bind to glutathione in the presence of ROS, especially βIII. In contrast, βI did not bind to glutathione. Our results suggest that βII and βIII, but especially βIII, may play an important role in the response of neuronal cells to stress. In view of the high levels of βII and βIII expressed in the nervous system it is conceivable that these tubulin isotypes may use their sulfhydryl groups to scavenge ROS and protect neuronal cells against oxidative stress.

Abstract 5105: Structure-activity relationship study of pyrrolo[2,3- d]pyrimidines as microtubule targeting antitumor agents

Abstract Microtubules are cytoskeleton protein polymers composed of αß-tubulin heterodimers which play pivotal roles in cell division, cellular transport and cell signaling. Interfering with microtubule dynamics is a well-established strategy for the treatment of cancer. However, clinical utility of microtubule targeting agents (MTAs) in cancer chemotherapy is often limited by the emergence of drug resistance. Expression of P-glycoprotein (Pgp) or the βIII isotype of tubulin, are two clinically established drug resistance mechanisms. We previously reported that compound AG346 binds to the colchicine site of tubulin and has microtubule depolymerization activity in A-10 cells (EC50 = 365 nM). Molecular modeling and docking studies of this compound in the X-ray crystal structure of tubulin (PDB: 402B) suggested that the pyrrole NH can be substituted with alkyl chains of optimal length to obtain an altered binding conformation which can facilitate interaction with the hydrophobic pocket formed by amino acid Val181 of side chain A, and Lys352 and Thr314 of side chain B. We designed and synthesized a variety of pyrrole NH-alkyl substituted compounds and determined that the compound AG473 shows a 43-fold improved microtubule depolymerization potency (EC50 = 8.4 nM) in A-10 cells, compared to the lead compound. In addition, this compound inhibited the growth of human melanoma cancer cell line MDA-MB-435 in culture with an IC50 of 7.2 nM, which is a 5-fold improvement in potency over the parent compound. These analogs also circumvented resistance mediated by Pgp and βIII-tubulin. These attributes provide the impetus for further preclinical development of these compounds as microtubule targeted antitumor agents in vivo and these studies are currently underway. Citation Format: Aleem Gangjee, Tasdique M. Quadery, Susan L. Mooberry. Structure-activity relationship study of pyrrolo[2,3- d]pyrimidines as microtubule targeting antitumor agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5105. doi:10.1158/1538-7445.AM2017-5105

Design, synthesis, and structure–activity relationships of pyrimido[4,5-b]indole-4-amines as microtubule depolymerizing agents that are effective against multidrug resistant cells

To identify the structural features of 9H-pyrimido[4,5-b]indoles as microtubule depolymerizers, pyrimido[4,5-b]indoles 2–8 with varied substituents at the 2-, 4- and 5-positions were designed and synthesized. Nucleophilic displacement of 2,5-substituted-4-chloro-pyrimido[4,5-b]indoles with appropriate arylamines was the final step employed in the synthesis of target compounds 2–8. Compounds 2 and 6 had two-digit nanomolar potency (IC50) against MDA-MB-435, SK-OV-3 and HeLa cancer cells in vitro. Compounds 2 and 6 also depolymerized microtubules comparable to the lead compound 1. Compounds 2, 3, 6 and 8 were effective in cells expressing P-glycoprotein or the βIII isotype of tubulin, mechanisms that are associated with clinical drug resistance to microtubule targeting drugs. Proton NMR and molecular modeling studies were employed to identify the structural basis for the microtubule depolymerizing activity of pyrimido[4,5-b]indoles.

Mobility and Core-Protein Binding Patterns of Disordered C-Terminal Tails in β-Tubulin Isotypes.

Although they play a significant part in the regulation of microtubule structure, dynamics, and function, the disordered C-terminal tails of tubulin remain invisible to experimental structural methods and do not appear in the crystallographic structures that are currently available in the Protein Data Bank. Interestingly, these tails concentrate most of the sequence variability between tubulin isotypes and are the sites of the principal post-translational modifications undergone by this protein. Using homology modeling, we developed two complete models for the human αI/βI- and αI/βIII-tubulin isotypes that include their C-terminal tails. We then investigated the conformational variability of the two β-tails using long time-scale classical molecular dynamics simulations that revealed similar features, notably the unexpected presence of common anchoring regions on the surface of the tuulin dimer, but also distinctive mobility or interaction patterns, some of which could be related to the tail lengths and charge distributions. We also observed in our simulations that the C-terminal tail from the βI isotype, but not the βIII isotype, formed contacts in the putative binding site of a recently discovered peptide that disrupts microtubule formation in glioma cells. Hindering the binding site in the βI isotype would be consistent with this peptide's preferential disruption of microtubule formation in glioma, whose cells overexpress βIII, compared to normal glial cells. While these observations need to be confirmed with more intensive sampling, our study opens new perspectives for the development of isotype-specific chemotherapy drugs.

Structure-based approaches for the design of benzimidazole-2-carbamate derivatives as tubulin polymerization inhibitors.

Microtubules are highly dynamic assemblies of α/β-tubulin heterodimers whose polymerization inhibition is among one of the most successful approaches for anticancer drug development. Overexpression of the class I (βI) and class III (βIII) β-tubulin isotypes in breast and lung cancers and the highly expressed class VI (βVI) β-tubulin isotype in normal blood cells have increased the interest for designing specific tubulin-binding anticancer therapies. To this end, we employed our previously proposed model of the β-tubulin-nocodazole complex, supported by the recently determined X-ray structure, to identify the fundamental structural differences between β-tubulin isotypes. Moreover, we employed docking and molecular dynamics (MD) simulations to determine the binding mode of a series of benzimidazole-2-carbamete (BzC) derivatives in the βI-, βIII-, and βVI-tubulin isotypes. Our results demonstrate that Ala198 in the βVI isotype reduces the affinity of BzCs, explaining the low bone marrow toxicity for nocodazole. Additionally, no significant differences in the binding modes between βI- and βIII-BzC complexes were observed; however, Ser239 in the βIII isotype might be associated with the low affinity of BzCs to this isotype. Finally, our study provides insight into the β-tubulin-BzC interaction features essential for the development of more selective and less toxic anticancer therapeutics.

Novel mutations involving βI-, βIIA-, or βIVB-tubulin isotypes with functional resemblance to βIII-tubulin in breast cancer.

Tubulin is the target for very widely used anti-tumor drugs, including Vinca alkaloids, taxanes, and epothilones, which are an important component of chemotherapy in breast cancer and other malignancies. Paclitaxel and other tubulin-targeting drugs bind to the β subunit of tubulin, which is a heterodimer of α and β subunits. β-Tubulin exists in the form of multiple isotypes, which are differentially expressed in normal and neoplastic cells and differ in their ability to bind to drugs. Among them, the βIII isotype is overexpressed in many aggressive and metastatic cancers and may serve as a prognostic marker in certain types of cancer. The underpinning mechanisms accounting for the overexpression of this isotype in cancer cells are unclear. To better understand the role of β-tubulin isotypes in cancer, we analyzed over 1000 clones from 90 breast cancer patients, sequencing their β-tubulin isotypes, in search of novel mutations. We have elucidated two putative emerging molecular subgroups of invasive breast cancer, each of which involve mutations in the βI-, βIIA-, or βIVB isotypes of tubulin that increase their structural, and possibly functional, resemblance to the βIII isotype. A unifying feature of the first of the two subgroups is the mutation of the highly reactive C239 residue of βI- or βIVB-tubulin to L239, R239, Y239, or P239, culminating in probable conversion of these isotypes from ROS-sensitive to ROS-resistant species. In the second subgroup, βI, βIIA, and βIVB have up to seven mutations to the corresponding residues in βIII-tubulin. Given that βIII-tubulin has emerged as a pro-survival factor, overexpression of this isotype may confer survival advantages to certain cancer cell types. In this mini-review, we bring attention to a novel mechanism by which cancer cells may undergo adaptive mutational changes involving alternate β-tubulin isotypes to make them acquire some of the pro-survival properties of βIII-tubulin. These "hybrid" tubulins, combining the sequences and/or properties of two wild-type tubulins (βIII and either βI, βIIA, or βIVB), are novel isotypes expressed solely in cancer cells and may contribute to the molecular understanding and stratification of invasive breast cancer and provide novel molecular targets for rational drug development.

Abstract P6-13-07: The taccalonolides are novel microtubule stabilizers that covalently bind tubulin and have in vivo efficacy in drug resistant tumors

Abstract Some of the most effective drugs used in the treatment of breast cancer are microtubule stabilizers. However, there are limitations to their clinical efficacy, including inherent and acquired drug resistance. All microtubule stabilizers that are currently approved for clinical use bind within the taxane pocket on β-tubulin in a reversible manner. The taccalonolides are a novel class of microtubule stabilizers that have a similar profile of microtubule stabilization as the taxanes, but circumvent drug resistance mediated by expression of drug efflux pumps, mutations in the taxane binding site, or overexpression of the βIII isotype of tubulin. We have shown that one important difference between the taccalonolides and clinically approved microtubule stabilizers is that the taccalonolides form a covalent bond to β-tubulin. This distinct interaction allows for irreversible binding, which explains their ability to avoid drug efflux mechanisms and likely belies their exquisite potency in in vivo antitumor models which allows for delivery in aqueous solvents. Serum stability and binding studies, microsomal clearance and pharmacokinetic analysis were performed with both taccalonolides AF and AJ to more fully understand the properties of this class of compounds. We found that both taccalonolides had low microsomal intrinsic clearance rates with no evidence of serum binding and had half-lives similar to paclitaxel in vivo. Like other microtubule targeted agents, taccalonolide AF has a narrow therapeutic window with antitumor effects accompanied by body weight loss. Interestingly, direct injection of taccalonolide AF into a xenograft tumor was highly effective with no associated toxicities at low doses, indicating that targeted delivery to the tumor would greatly increase the efficacy and decrease toxicities. To this end, efforts to promote the targeted delivery of taccalonolide AF to the tumor are being evaluated. Citation Format: Risinger AL, Li J, Benavides R, Kuhn JG, Mooberry SL. The taccalonolides are novel microtubule stabilizers that covalently bind tubulin and have in vivo efficacy in drug resistant tumors. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-13-07.

Biological Characterization of an Improved Pyrrole-Based Colchicine Site Agent Identified through Structure-Based Design.

A refined model of the colchicine site on tubulin was used to design an improved analog of the pyrrole parent compound, JG-03-14. The optimized compound, NT-7-16, was evaluated in biological assays that confirm that it has potent activities as a new colchicine site microtubule depolymerizer. NT-7-16 exhibits antiproliferative and cytotoxic activities against multiple cancer cell lines, with IC(50) values of 10-16 nM, and it is able to overcome drug resistance mediated by the expression of P-glycoprotein and the βIII isotype of tubulin. NT-7-16 initiated the concentration-dependent loss of cellular microtubules and caused the formation of abnormal mitotic spindles, leading to mitotic accumulation. The direct interaction of NT-7-16 with purified tubulin was confirmed, and it was more potent than combretastatin A-4 in these assays. Binding studies verified that NT-7-16 binds to tubulin within the colchicine site. The antitumor effects of NT-7-16 were evaluated in an MDA-MB-435 xenograft model and it had excellent activity at concentrations that were not toxic. A second compound, NT-9-21, which contains dichloro moieties in place of the 3,5-dibromo substituents of NT-7-16, had a poorer fit within the colchicine site as predicted by modeling and the Hydropathic INTeractions score. Biological evaluations showed that NT-9-21 has 10-fold lower potency than NT-7-16, confirming the modeling predictions. These studies highlight the value of the refined colchicine-site model and identify a new pyrrole-based colchicine-site agent with potent in vitro activities and promising in vivo antitumor actions.

Abstract 1665: Design, synthesis, biological evaluation of cyclopenta[d]pyrimidines as antitubulin agents and discovery of N-(4-methylthiophenyl) and N-(4-dimethylaminophenyl) substituted N,2-dimethyl-cyclopenta[d]pyrimidines as long acting and poten

Abstract Compounds that bind to the colchicine site of microtubules have promising antitumor potential. Some of these agents also possess the ability to overcome multiple drug resistance mechanisms, including those mediated by the expression of P-glycoprotein (Pgp) or expression of the tubulin β-III isotype. We have reported that compounds 1 and 2 were potent antiproliferative agents that bind to the colchicine site of tubulin. The previous structure-activity relationship studies of cyclopenta[d]pyrimidine based antitubulin agents demonstrated the importance of the para-methoxyphenyl moiety for antiproliferative activity. However, the literature suggests that the major metabolism of the methoxyphenyl moiety was likely O-demethylation. In order to increase the potency and in vivo stability of 1, we focused our attention on the variation of the para-methoxy aniline moiety. Compound 3 with a 4′-methylthiophenyl substitution was designed to decrease potential metabolic demethylation. This S-methyl analog was a potent inhibitor of proliferation with an IC50 of 4.6 ± 0.5 nM in MDA-MB-435 cells. The possible sulfoxide metabolite, 4 was synthesized and had an IC50 = 7.9 ± 0.5 nM. Each of these depolymerized microtubules. In addition, the 4′-dimethylamino moiety compound 5 also demonstrated potent antitubulin and antiproliferative activity (MDA-MB-435: IC50 = 8.2 ± 0.6 nM). The possible N-demethylated metabolite of 5, para-methylamino 6 was also a highly potent antiproliferative agent (MDA-MB-435: IC50 = 20 ± 1.9 nM). The parent compounds 3 and 5, as well as their probable metabolites 4 and 6, inhibited the binding of [3H]colchicine to tubulin, which showed that these compounds bind to the colchicine site. The potent antiproliferative activities of the parent compounds and the potential metabolites of compounds 3 and 5 suggest the utility of 3 and 5 as long acting, potent antimitotic agents. Finally, the in vivo efficacy of the S-methyl analog 2 was demonstrated against MDA-MB-435 triple negative breast cancer murine xenograft model, indicating the potential utility of these analogs as antitumor agents. Citation Format: Aleem Gangjee, Weiguo Xiang, Susan L. Mooberry, Ernest Hamel. Design, synthesis, biological evaluation of cyclopenta[d]pyrimidines as antitubulin agents and discovery of N-(4-methylthiophenyl) and N-(4-dimethylaminophenyl) substituted N,2-dimethyl-cyclopenta[d]pyrimidines as long acting and poten [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1665. doi:10.1158/1538-7445.AM2015-1665

Designing and Testing of Novel Taxanes to Probe the Highly Complex Mechanisms by Which Taxanes Bind to Microtubules and Cause Cytotoxicity to Cancer Cells

Our previous work identified an intermediate binding site for taxanes in the microtubule nanopore. The goal of this study was to test derivatives of paclitaxel designed to bind to this intermediate site differentially depending on the isotype of β-tubulin. Since β-tubulin isotypes have tissue-dependent expression—specifically, the βIII isotype is very abundant in aggressive tumors and much less common in normal tissues—this is expected to lead to tubulin targeted drugs that are more efficacious and have less side effects. Seven derivatives of paclitaxel were designed and four of these were amenable for synthesis in sufficient purity and yield for further testing in breast cancer model cell lines. None of the derivatives studied were superior to currently used taxanes, however computer simulations provided insights into the activity of the derivatives. Our results suggest that neither binding to the intermediate binding site nor the final binding site is sufficient to explain the activities of the derivative taxanes studied. These findings highlight the need to iteratively improve on the design of taxanes based on their activity in model systems. Knowledge gained on the ability of the engineered drugs to bind to targets and bring about activity in a predictable manner is a step towards personalizing therapies.

Abstract 836: Potent antitubulin and antitumor activities influenced by the 3-D conformational shape of bicyclic fused pyrimidines

Abstract Compounds that bind to the tubulin colchicine site cause microtubule depolymerization, inhibit the polymerization of purified tubulin and cause cancer cells to accumulate in mitosis. Some colchicine-site agents additionally have the ability to overcome multiple drug resistance mechanisms, including those mediated by the expression of P-glycoprotein (Pgp) or expression of the β-III isotype of tubulin. We have reported a series of aniline substituted pyrimidines and fused bicyclic and tricyclic pyrimidine compounds that bind to the colchicine site. These compounds are single to triple digit nanomolar inhibitors of cancer cell proliferation. We hypothesized that the conformational orientation of the 4-aniline and the fused pyrimidine scaffold plays an important role in the biological activity of these compounds. In this study, we utilized NMR (NOESY) and molecular modeling to compare the lowest energy conformation of different 4-substituted 2-methyl cyclopenta[d]pyrimidines and their microtubule depolymerizing, colchicine site binding and antiproliferative activities. Among the 4-position substituents were N-methyl-4’-methoxynaphthyl-1’-amino (1), N-methyl-6’-methoxynaphthyl-1’-amino (2) and N-methyl-5’-methoxy-2’-amino (3). The IC50 values towards the MDA-MB-435 tumor cell line were 250 nM for 1 and 10 nM for 3. NMR (NOESY) demonstrated that compound 2 lost the free rotation of the naphthyl ring attached to the cyclopenta[d]pyrimidine ring due to 5-H steric hindrance and did not cause microtubule depolymerization. Compounds 1 and 3 retain the ability to rotate the naphthyl ring even though they adopt slightly different stable conformers. The stable conformational differences of these three compounds afford different binding poses in docking to the colchicine site on tubulin (PDB: 1SA0, MOE 2012), which explains the significant differences in antiproliferative activity, microtubule depolymerizing and inhibition of colchicine binding. This study affords a lead compound that defines a 104o (orthogonal) orientation of the naphthyl ring to the cyclopenta[d]pyrimidine scaffold for the best biological activities. Citation Format: Aleem Gangjee, Weiguo Xiang, Susan L. Mooberry, Ernest Hamel. Potent antitubulin and antitumor activities influenced by the 3-D conformational shape of bicyclic fused pyrimidines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 836. doi:10.1158/1538-7445.AM2014-836

Synthesis of N4-(substituted phenyl)-N4-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines and identification of new microtubule disrupting compounds that are effective against multidrug resistant cells

A series of fourteen N4-(substituted phenyl)-N4-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cyclization of 2-amino-6-hydrazinylpyrimidin-4(3H)-one with cyclohexanone, followed by oxidation, chlorination and displacement with appropriate anilines. Compounds 6, 14 and 15 had low nanomolar potency against MDA-MB-435 tumor cells and depolymerized microtubules. Compound 6 additionally had nanomolar GI50 values against 57 of the NCI 60-tumor panel cell lines. Mechanistic studies showed that 6 inhibited tubulin polymerization and [3H]colchicine binding to tubulin. The most potent compounds were all effective in cells expressing P-glycoprotein or the βIII isotype of tubulin, which have been associated with clinical drug resistance. Modeling studies provided the potential interactions of 6, 14 and 15 within the colchicine site.

Rationalization of paclitaxel insensitivity of yeast β-tubulin and human βIII-tubulin isotype using principal component analysis

BackgroundThe chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among β-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin, within a common theoretical framework, we have performed structural principal component analyses of β-tubulin sequences across eukaryotes.ResultsThe paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of β-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in βIII isotype was shown to be crucial for paclitaxel-resistance.ConclusionsThe present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human βIII tubulin isotype, through two common collective sequence vectors.

Tubulins as Therapeutic Targets in Cancer: from Bench to Bedside

Tubulin is the target of some of the most widely used and time-honored anticancer tubulin-binding agents (TBAs). The clinical usefulness of many TBAs has been held back as a result of tumor cell drug-resistance. The elucidation of the three-dimensional structure of αβ-tubulin dimer has provided an opportunity for rational drug design aimed at generating compounds that will target tubulin in therapeutically more efficacious ways compared to presently available drugs. An issue to be addressed is which one(s) of the tubulin species, their isotypes, or their posttranslationally modified forms, should be specifically targeted in cancer chemotherapy. This review offers a critical appraisal of current knowledge on tubulins in cancer and an update on new anti-neoplastic microtubule-targeted treatment strategies. Specifically, it examines, across disciplines, cellular/molecular, biochemical, clinical/pathological, and pharmacological aspects of β-tubulin isotypes, posttranslational modifications of tubulin dimers, γ-tubulin and microtubule nucleation, and microtubule regulatory proteins. Emphasis is placed on the overexpression of (i) the βIII isotype, which functions as a survival factor associated with dynamic instability of microtubules; (ii) γ-tubulin, a key microtubule nucleating protein; and (iii) the microtubule severing enzyme spastin, involved in cell motility and proliferation of glioblastoma cells. The role of βIII-tubulin in resistance of cancer cells to taxanes is examined. Attention is called to the novel concept that βIII-tubulin functions as a "gateway" for prosurvival signals in partnership with GTPases, such as GBP1. Appraisal is also offered on epothilones and the concept of hypersensitization to TBAs as promising therapeutic strategies in taxane resistant epithelial cancers and in high-grade gliomas.