Indira Padmalayam

KIFC1 is a novel potential therapeutic target for breast cancer.

Kinesin-like protein KIFC1, a normally nonessential kinesin motor, plays a critical role in centrosome clustering in cancer cells and is essential for the survival of cancer cells. Herein, we reported that KIFC1 expression is up-regulated in breast cancer, particularly in estrogen receptor negative, progesterone receptor negative and triple negative breast cancer, and is not associated with epidermal growth factor receptor 2 status. In addition, KIFC1 is highly expressed in all 8 tested human breast cancer cell lines, but is absent in normal human mammary epithelial cells and weakly expressed in 2 human lung fibroblast lines. Moreover, KIFC1 silencing significantly reduced breast cancer cell viability. Finally, we found that PJ34, a potent small molecule inhibitor of poly(ADP-ribose) polymerase, suppressed KIFC1 expression and induced multipolar spindle formation in breast cancer cells, and inhibited cell viability and colony formation within the same concentration range, suggesting that KIFC1 suppression by PJ34 contributes to its anti-breast cancer activity. Together, these results suggest that KIFC1 is a novel promising therapeutic target for breast cancer.

A Simple and Specific Method for Estimation of Lipoic Acid in Human Plasma by High Performance Liquid Chromatography

A rapid high performance liquid chromatographic method for determination of lipoic acid in human plasma was developed and validated. The method involved extraction of lipoic acid in ethanol consisting of 50 mM disodium hydrogen phosphate: acetonitrile: methanol in the ratio of 50:30:20. The separation was done using a C18 column (150 mm) and detection was carried out using UV detection at 201 nm. The assay was found to be linear in the range of 0.78-50 μg/ml with the correlation coefficient of 0.9998. Intra and inter-day variations were determined by processing each standard concentration in duplicate for five consecutive days. The average recovery of lipoic acid from plasma was 113%. The developed method demonstrates better sensitivity, precision, accuracy, stability and linearity when compared with the methods previously used. The method is simple and can be used for the determination of lipoic acid in basic research studies as well as in standard clinical laboratories.

Discovery of a novel inhibitor of kinesin-like protein KIFC1

Historically, drugs used in the treatment of cancers also tend to cause damage to healthy cells while affecting cancer cells. Therefore, the identification of novel agents that act specifically against cancer cells remains a high priority in the search for new therapies. In contrast with normal cells, most cancer cells contain multiple centrosomes which are associated with genome instability and tumorigenesis. Cancer cells can avoid multipolar mitosis, which can cause cell death, by clustering the extra centrosomes into two spindle poles, thereby enabling bipolar division. Kinesin-like protein KIFC1 plays a critical role in centrosome clustering in cancer cells, but is not essential for normal cells. Therefore, targeting KIFC1 may provide novel insight into selective killing of cancer cells. In the present study, we identified a small-molecule KIFC1 inhibitor, SR31527, which inhibited microtubule (MT)-stimulated KIFC1 ATPase activity with an IC50 value of 6.6 μM. By using bio layer interferometry technology, we further demonstrated that SR31527 bound directly to KIFC1 with high affinity (Kd=25.4 nM). Our results from computational modelling and saturation-transfer difference (STD)-NMR experiments suggest that SR31527 bound to a novel allosteric site of KIFC1 that appears suitable for developing selective inhibitors of KIFC1. Importantly, SR31527 prevented bipolar clustering of extra centrosomes in triple negative breast cancer (TNBC) cells and significantly reduced TNBC cell colony formation and viability, but was less toxic to normal fibroblasts. Therefore, SR31527 provides a valuable tool for studying the biological function of KIFC1 and serves as a potential lead for the development of novel therapeutic agents for breast cancer treatment.

3D Cell Cultures: Mimicking In Vivo Tissues for Improved Predictability in Drug Discovery

Abstract The use of 2D cell culture has been the mainstay of cell biology and has been applied to all of our modern day drug discovery and development activities. However, the identification of new therapeutics has not achieved the success rates anticipated. One potential explanation is the complexity and lack of predictability of our current research methods, including 2D cell culture. Numerous studies have indicated that cells grown in a flat environment lose critical parameters and thus could provide false information when used in a drug discovery paradigm. In this chapter we describe the advances in 3D cell culture and the potential applications to drug discovery. Highlighted are the differences between cells grown in 2D versus 3D with respect to cell physiology and their responses to external stimuli. These differences can have profound effects on the type of compounds identified and prioritized for further research or development.

Identification of the binding site of an allosteric ligand using STD-NMR, docking, and CORCEMA-ST calculations

Singling out the truth: A combined application of STD-NMR, molecular docking, and CORCEMA-ST calculations is described as an attractive, easily applicable tool for the identification and validation of the binding site for allosteric ligands, with potential application as an aid in drug discovery research.

3D Cell Cultures

Abstract The use of 2D cell culture has been the mainstay of cell biology and has been applied to all of our modern day drug discovery and development activities. However, the identification of new therapeutics has not achieved the success rates anticipated. One potential explanation is the complexity and lack of predictability of our current research methods, including 2D cell culture. Numerous studies have indicated that cells grown in a flat environment lose critical parameters and thus could provide false information when used in a drug discovery paradigm. In this chapter we describe the advances in 3D cell culture and the potential applications to drug discovery. Highlighted are the differences between cells grown in 2D versus 3D with respect to cell physiology and their responses to external stimuli. These differences can have profound effects on the type of compounds identified and prioritized for further research or development.

Association of A1538G and C2437T Single Nucleotide Polymorphisms in Heat Shock Protein 70 Genes with Type 2 Diabetes

Objective: To examine 2 single nucleotide polymorphisms (SNPs) of the heat shock protein 70-2 (HSPA1B ) and 70-hom (HSPA1L) genes in patients with type 2 diabetes mellitus (T2DM) in our study population. Methods: A total of 104 patients with T2DM and 124 healthy control individuals were included in the study. G1538A in HSPA1B and C2437T in HSPA1L polymorphisms were determined using the polymerase chain reaction–restriction fragment length polymorphism technique. Results: The frequency of the HSPA1B genotype was as follows: AA: 33 (26.6%); AG: 82 (66.2%); and GG: 9 (7.2%) in the control group and AA: 2 (2.0%), AG: 57 (54.8%), and GG: 45 (43.2%) in the T2DM group (P<.001). The frequency of the HSPA1L genotype was as follows: CC: 20 (16.1%), CT: 92 (74.2%), and TT: 12 (9.7%) in the control group and CC: 8 (7.6%), CT: 65 (62.5%), and TT: 31 (29.8%) in the T2DM group (P = .003). Conclusion: Our findings indicate that HSPA1B, HSPA1L, and their functional polymorphisms may play a role in the pathogenesis of T2DM.

Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode

Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein.

Discovery of Novel Allosteric Eg5 Inhibitors Through Structure-Based Virtual Screening

Mitotic kinesin Eg5 is an attractive anticancer drug target. Discovery of Eg5 inhibitors has been focused on targeting the ‘monastrol‐binding site’. However, acquired drug resistance has been reported for such inhibitors. Therefore, identifying new Eg5 inhibitors which function through a different mechanism(s) could complement current drug candidates and improve drug efficacy. In this study, we explored a novel allosteric site of Eg5 and identified new Eg5 inhibitors through structure‐based virtual screening. Experiments with the saturation‐transfer difference NMR demonstrated that the identified Eg5 inhibitor SRI35566 binds directly to Eg5 without involving microtubules. Moreover, SRI35566 and its two analogs significantly induced monopolar spindle formation in colorectal cancer HCT116 cells and suppressed cancer cell viability and colony formation. Together, our findings reveal a new allosteric regulation mechanism of Eg5 and a novel drug targeting site for cancer therapy.

Abstract LB-055: High-throughput screening efforts for the identification of selective and potent inhibitors of CD38 for the treatment of hematological cancers

By means of a phenomenon termed “the Warburg effect,” tumor cells shift their energy production by mitochondrial oxidative phosphorylation to aerobic glycolysis, resulting in the upregulation of glucose consumption and increased cellular oxidative and nitrosative stress. To compensate for such toxic levels of ROS/RNS, cancer cells rely heavily on their antioxidant defense mechanisms, which are largely controlled by the NAD(P)/NAD(P)H redox partners. We found that the modulation of NAD metabolism in vivo, specifically via the deletion of the NAD glycohydrolase CD38, resulted in increased production of intrinsic ROS and increased DNA damage following exposure to chemotherapeutics. Furthermore, in vitro experiments showed that CD38 knockdown in CD38-expressing tumor cells prevented the generation of stable transfectants, highlighting a role for CD38 in tumor cell survival. In light of these findings, we hypothesized that pharmacological inhibition of CD38 may be an effective therapy for the treatment of hematological cancers, in particular those which uniformly overexpress CD38, such as MM and chronic lymphocytic leukemia. Indeed, treatment of human MM cell lines LP-1 and KMS-12-PE with CD38 antagonists sensitized the cells to standard ROS-inducing chemotherapeutics. We conducted a high-throughput screening (HTS) campaign of over two hundred thousand unique and non-proprietary lead-like compounds using an optimized and miniaturized HTS based on a luminescent NAD quantitation platform. Five hundred active hits were analyzed for toxicity using a cell-based HTS assay purposely designed with CD38-negative HEK293 cells to avoid elimination of desirable compounds toxic to CD38-positive cells. Hits with non-specific properties, such as PAINS (Pan Assay Interference Compounds), were removed by computational filtering, and the remaining compounds were tested for inhibition of human CD38 activity in cells. The last phase of the compound progression pathway involved testing for non-selective inhibition of other NAD-consuming enzymes, namely Poly(ADP-ribose) polymerase-1, and Sirtuin-1, which led to the identification of two distinct chemical series that exhibit >10-fold selectivity for human CD38. Hit-to-lead chemistry is currently underway to synthesize key analogs by rational drug design. In summary, our data suggests that CD38 is an antioxidant protein selectively used to maintain a cellular redox balance, and proposes that targeting the enzymatic activity of CD38 may be a novel therapeutic strategy for chemosensitizing hematological cancers. Our HTS campaign efforts are paving the way for the discovery and development of potent and selective small-molecule inhibitors of CD38. Citation Format: Davide Botta, Tulin Dadali, Betty J. Mousseau, Fen Zhou, Michael D. Schultz, Esther Zumaquero, Anna Manouvakhova, Melinda I. Sosa, Sara N. McKellip, LaKeisha Woods, Nichole A. Tower, Larry J. Ross, Lynn Rasmussen, E. Lucille White, Indira Padmalayam, Wei Zhang, Maaike Everts, Corinne E. Augelli-Szafran, James R. Bostwick, Mark J. Suto, Frances E. Lund. High-throughput screening efforts for the identification of selective and potent inhibitors of CD38 for the treatment of hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-055.