Identification and characterization of an irreversible inhibitor of CDK2

Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)- 9H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds

Computational Design, Synthesis, Characterization and Pharmacological Evaluation of Some Piperidine Derivatives

The Aurora kinase family is a collection of highly related serine/threonine kinases that functions as a key regulator of mitosis. In mammalian cells, Aurora has evolved into three related kinases known as Aurora-A, Aurora-B, and Aurora-C. These kinases are over expressed in a number of human cancers, and transfection studies have established Aurora-A as a bone fide oncogene. Because Aurora over expression is associated with malignancy, these kinases have been targeted for cancer therapy. So in the present study, it was decided to design some inhibitory lead compounds of Aurora kinase A as using computational tools like Catalyst (pharmacophore modeling) and GLIDE(structure based drug design/Docking). Then the designed compounds were synthesized and screened for anticancer studies. Eighty two Aurora A kinase inhibitors from Medicinal Chemistry Journals were selected for modeling studies based on chemical and biological diversity. The selected molecules were then divided into 21 training set molecules and 61 test set molecules. Using the training set molecules pharmacophore models (hypothesis) were generated in Hypogen (Catalyst). The most active molecule in the training set fits very well with the top scoring pharmacophore hypothesis. The best hypothesis consists of one hydrogen bond acceptor, one hydrophobic aliphatic and two ring aromatics. The best hypothesis Hypo-1 is characterized by the highest cost difference (58 bits), lowest RMS deviation (1.30) with a correlation of 0.94. The best pharmacophore hypothesis was used to screen the 61 Aurora A Kinase inhibitors in the Aurora kinase inhibitor data base. The model developed was shown to be a good model with 0.65 as Goodness of Hit score (GH) and a enrichment factor of 1.154. GLIDE was the docking program used for the structure based drug design. The 82 Aurora A inhibitors used for the pharmacophore studies were considered for docking study to develop comparative model. Out of the 82 inhibitors 21 were used in the training set. Crystal structure of Aurora A (PDB code: Imq4) was employed for the docking studies Structure based docking studies were carried out using Glide on Aurora A kinase inhibitors to the 3D structure of Aurora A kinase and generated 50 best docking poses. The best poses were selected based on the scoring functions and poses orientation with the active site amino acids. To get a better VS model, a MLR analysis was carried out using pharmacophore model and the docking scoring function. A combination of Pharmacophore model, GLIDE SP dock score gave a good model. The VS model obtained was further used to search the virtual library consisting of 10,000 structurally diversified molecules generated using fragment and knowledge based design, which yielded 300 molecules as potent Hits. The hits obtained were used for PASS prediction studies. A consensus was obtained between the docking scores and the PASS prediction values and finally 40 Hits were selected for synthesis and screening. The docking scores >-7 and prediction values above 0.6 were taken into consideration. PASS predicted some of the molecules to be active against colorectal cancer (1A-29A) and some other molecules to be inhibitors of phosphatase enzyme (P1-P11). A Drug likeness screening was carried out for all the 40 Hits including Lipinski rule of five and Toxicity assessment. All most all the compounds exhibited 2 violations of the Lipinski rule which was found to be normal with anticancer drugs. It is proven that kinase inhibitors in general have high molecular weight and LogP values. So these violations were accepted in the present study. In toxicity assessment all the compounds showed Green colour code except nine molecules having nitro and dimethyl amino substitution exhibiting mutagenicity and Tumorigenicity. But still these molecules were also included in the in vitro screening because so many active scaffolds have these substructures and their activities were also predicted to be good. These hits can be further refined to reduce the unwanted reactions by including detoxifying sub structures. The designed molecules have piperidine-4-ones scaffold attached with 2-Aminopyrimidine (1A-29A) and 2-Pyrazoline (P1-P11) substructures. The Schiff bases and Mannich bases of piperidine-4-ones were synthesized according to the synthetic scheme and characterized by IR,1H NMR,13 C NMR, COSY NMR and Mass spectroscopy. The compounds were subjected to in vitro anticancer studies in colorectal cell lines (1A-29A).The compounds 21A and 25A show the least IC50 values 0.01 and 0.01 respectively. The compound 21A maintains the same level of activity through out the working range (0.01-100 μM). There is no concentration related gradation in the activity profile. In the case of compound 25A, the peak activity is noted in the minimum concentration of 0.01 μM itself. Even though there is a decrease in the activity with increase in the concentration, the activity profile remained well above the required level. The other compounds showing significant activity are 3A, 10A, 13A and 26A. The compounds 6A, 9A and 16A show less activity with IC50 values in the range of 10 μM. All the other compounds showed moderate activity with IC50 values in the range of 1-5 μM. The compounds P1-P11 were subjected to phosphatase inhibition activity. The compounds P1-P11 were used in three concentration levels (50/125/250mcg). The absorbances of these solutions were measured at 620nm after carrying out the assay with Folin’s reagent. The concentrations of the phenol formed in these solutions were obtained from the standard graph of phenol. From the analysis of the data it can be seen that all the compounds P1-P11 are possessing phosphatase inhibition activity. In addition to that they also show a gradation in their dose response. All the compounds show less inhibition at 50 mcg, moderate inhibition at 125 mcg and a fairly good inhibition at 250 mcg. The compounds P1-P11 have 2-pyrazoline moiety as a sub structure in common. The compounds P1-P5 at 50 mcg concentration level shows very less inhibition. A concentration dependent increase in inhibitory activity was observed with P1, P2, P3, P4, and P5. That is better inhibitory activity was observed with higher concentrations (250 mcg). The compounds P6-P11 showed better inhibition in all the three concentration levels. In all these compounds the C3 of piperidin-4-one ring contain isopropyl group which may contribute to the increase in inhibitory activity. Of all compounds, the compound P3 at the 250mcg concentration level shows best activity. Thus the compounds P3, P7 and P11 can be further developed to get effective phosphatase inhibitors. By arriving at the leads 21A and 25A for anticancer activity, the aim of the work to develop leads for anticancer activity was fulfilled. These analogues can be novel templates for lead optimization purpose in cancer chemotherapy. To conclude the anticancer leads obtained in this study can be refined further to get a potent anticancer molecules. Drugs targeting multiple kinases have proven to be effective against treatment of various diseases. The activities of serine/threonine protein phosphatases needs further study, but it is clear that these enzymes are potential targets for novel therapeutics with applications in many diseases, including cancer, inflammatory diseases and neuro degeneration. Computational techniques have provided starting points for designing multiple inhibitors against individual targets using crystal structural information of kinases and pharmacophore of kinase inhibitors. So these techniques can be explored further to design new drug candidates for various diseases

In-silico investigation of Coenzyme A selectivity for Aurora A kinase and development of an Aurora A kinase-selective inhibitor as a potential anticancer agent

Centrosome amplification has been observed in most cancer cells, and is considered to be a “hallmark” of cancer cells. This process is commonly associated with chromosome segregation process in the mitosis phase in the cell cycle, which is tightly controlled by mitotic kinases. Among these kinases, the Aurora kinase family, Aurora A (AURKA), Aurora B (AURKB) and Aurora C (AURKC), ensures the accurate progression of mitosis, including the formation of a bipolar mitotic spindle, accurate segregation of chromosomes and the completion of cytokinesis. AURKA has been seen to have the largest role in mitotic progression and checkpoint control pathways and overexpression of AURKA is most associated with cancer. Hence, interfering with AURKA activity has been considered to be a promising approach to anticancer agents. Professor Gout’s group has recently shown that Coenzyme A (CoA) selectively inhibits AURKA activity (IC50 of 4.4 μM). However, its large molecular weight (>500) and negatively charged phosphate group make it unsuitable as a drug candidate. This project was set to investigate the possible binding modes of CoA in the catalytic domain of AURKA. The corresponding interactions between CoA and protein residues would provide some insights in the selectivity of CoA towards AURKA. Furthermore, based on the understanding of the interactions between CoA and the catalytic domain of AURKA and the possible reasons behind the selectivity of CoA towards AURKA, in silico design and synthesis of a new highly selective and potent AURKA inhibitor based on the structure of CoA and current lead compounds which are in clinical trials for Aurora kinase inhibitors could be carried out

Improving treatment of glioblastoma: new insights in targeting cancer stem cells effectively

Glioblastoma is the most common primary malignant brain tumour in the adult population. Despite multimodality treatment with surgery, radiotherapy and chemotherapy, outcomes are very poor, with less than 15% of patients alive after two years. Increasing evidence suggests that glioblastoma stem cells (GSCs) are likely to play an important role in the biology of this disease and are involved in treatment resistance and tumour recurrence following standard therapy. My thesis aims to address two main aspects of this research area: 1) optimization of methods to evaluate treatment responses of GSCs and their differentiated counterparts (non-GSCs), with a particular focus on a tissue culture model that resembles more closely the tumoral niche; 2) characterization of cell division and centrosome cycle of GSCs, investigating possible differences between these cells and non-GSCs, that would allow the identification of targets for new therapeutic strategies against glioblastomas. In the first part of my project, I optimized a clonogenic survival assay, to compare sensitivity of GSCs and non-GSCs to various treatments, and I developed the use of a 3-dimentional tissue culture system, that allows analysis of features and radiation responses of these two subpopulations in the presence of specific microenvironmental factors from the tumoral niche. In the second part, I show that GSCs display mitotic spindle abnormalities more frequently than non-GSCs and that they have distinctive features with regards to the centrosome cycle. I also demonstrate that GSCs are more sensitive than non-GSCs to subtle changes in Aurora kinase A activity, which result in a rapid increase in polyploidy and subsequently in senescence, with a consistent reduction in clonogenic survival. Based on these findings, I propose that kinases involved in the centrosome cycle need to be explored as a novel strategy to target GSCs effectively and improve outcomes of glioblastoma patients

Systems-Based Design of Bi-Ligand Inhibitors of Oxidoreductases: Filling the Chemical Proteomic Toolbox

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases

Carbohydrate-derived amphiphilic macromolecules: a biophysical structural characterization and analysis of binding behaviors to model membranes.

The design and synthesis of enhanced membrane-intercalating biomaterials for drug delivery or vascular membrane targeting is currently challenged by the lack of screening and prediction tools. The present work demonstrates the generation of a Quantitative Structural Activity Relationship model (QSAR) to make a priori predictions. Amphiphilic macromolecules (AMs) "stealth lipids" built on aldaric and uronic acids frameworks attached to poly(ethylene glycol) (PEG) polymer tails were developed to form self-assembling micelles. In the present study, a defined set of novel AM structures were investigated in terms of their binding to lipid membrane bilayers using Quartz Crystal Microbalance with Dissipation (QCM-D) experiments coupled with computational coarse-grained molecular dynamics (CG MD) and all-atom MD (AA MD) simulations. The CG MD simulations capture the insertion dynamics of the AM lipophilic backbones into the lipid bilayer with the PEGylated tail directed into bulk water. QCM-D measurements with Voigt viscoelastic model analysis enabled the quantitation of the mass gain and rate of interaction between the AM and the lipid bilayer surface. Thus, this study yielded insights about variations in the functional activity of AM materials with minute compositional or stereochemical differences based on membrane binding, which has translational potential for transplanting these materials in vivo. More broadly, it demonstrates an integrated computational-experimental approach, which can offer a promising strategy for the in silico design and screening of therapeutic candidate materials

Specificity rendering ‘hot-spots’ for aurora kinase inhibitor design: the role of non-covalent interactions and conformational transitions

The present study examines the conformational transitions occurring among the major structural motifs of Aurora kinase (AK) concomitant with the DFG-flip and deciphers the role of non-covalent interactions in rendering specificity. Multiple sequence alignment, docking and structural analysis of a repertoire of 56 crystal structures of AK from Protein Data Bank (PDB) has been carried out. The crystal structures were systematically categorized based on the conformational disposition of the DFG-loop [in (DI) 42, out (DO) 5 and out-up (DOU) 9], G-loop [extended (GE) 53 and folded (GF) 3] and αC-helix [in (CI) 42 and out (CO) 14]. The overlapping subsets on categorization show the inter-dependency among structural motifs. Therefore, the four distinct possibilities a) 2W1C (DI, CI, GE) b) 3E5A (DI, CI, GF) c) 3DJ6 (DI, CO, GF) d) 3UNZ (DOU, CO, GF) along with their co-crystals and apo-forms were subjected to molecular dynamics simulations of 40 ns each to evaluate the variations of individual residues and their impact on forming interactions. The non-covalent interactions formed by the 157 AK co-crystals with different regions of the binding site were initially studied with the docked complexes and structure interaction fingerprints. The frequency of the most prominent interactions was gauged in the AK inhibitors from PDB and the four representative conformations during 40 ns. Based on this study, seven major non-covalent interactions and their complementary sites in AK capable of rendering specificity have been prioritized for the design of different classes of inhibitors

Application of Small Epigenetic Modulators in Pediatric Medulloblastoma

Medulloblastoma is one of the most frequent among pediatric brain tumors, and it has been classified in various subgroups. Some of them already benefit from quite good therapeutic options, whereas others urgently need novel therapeutic approaches. Epigenetic modulators have long been studied in various types of cancer. Within this review, we summarize the main preclinical studies regarding epigenetic targets (such as HDAC, SIRT, BET, EZH2, G9a, LSD1, and DNMT) inhibitors in medulloblastoma. Furthermore, we shed light on the increasing number of applications of drug combinations as well as hybrid compounds involving epigenetic mechanisms. Nevertheless, in the studies published so far, mainly un-specific or old modulators have been used, and the PKs (brain permeability) have not been well-evaluated. Thus, these findings should be considered as a starting point for further improvement and not as a final result

Identification of Plk1 type II inhibitors by structure-based virtual screening

Protein kinases are targets for drug development. Dysregulation of kinase activity leads to various diseases, e.g. cancer, inflammation, diabetes. Human polo-like kinase 1 (Plk1), a serine/threonine kinase, is a cancer-relevant gene and a potential drug target which attracts increasing attention in the field of cancer therapy. Plk1 is a key player in mitosis and modulates entry into mitosis and the spindle checkpoint at the meta-/anaphase transition. Plk1 overexpression is observed in various human tumors, and it is a negative prognostic factor for cancer patients. The same catalytical mechanism and the same co-substrate (ATP) lead to the problem of inhibitor selectivity. A strategy to solve this problem is represented by targeting the inactive conformation of kinases. Kinases undergo conformational changes between active and inactive conformation and thus an additional hydrophobic pocket is created in the inactive conformation where the surrounding amino acids are less conserved. A "homology model" of the inactive conformation of Plk1 was constructed, as the crystal structure in its inactive conformation is unknown. A crystal structure of Aurora A kinase served as template structure. With this homology model a receptor-based pharmacophore search was performed using SYBYL7.3 software. The raw hits were filtered using physico-chemical properties. The resulting hits were docked using Gold3.2 software, and 13 candidates for biological testing were manually selected. Three compounds of the 13 tested exhibit anti-proliferative effects in HeLa cancer cells. The most potent inhibitor, SBE13, was further tested in various other cancer cell lines of different origins and displayed EC50 values between 12 microM and 39 microM. Cancer cells incubated with SBE13 showed induction of apoptosis, detected by PARP (Poly-Adenosyl-Ribose-Polymerase) cleavage, caspase 9 activation and DAPI staining of apoptotic nuclei