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Exploring the Pi3ka and G Binding Sites by Homology Modeling And

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Exploring the Pi3ka and G Binding Sites by Homology Modeling And U UNIVERSITY OF CINCINNATI Date: I, , hereby submit this original work as part of the requirements for the degree of: in It is entitled: Student Signature: This work and its defense approved by: Committee Chair: Approval of the electronic document: I have reviewed the Thesis/Dissertation in its final electronic format and certify that it is an accurate copy of the document reviewed and approved by the committee. Committee Chair signature: Exploring the PI3K and binding sites by homology modeling and inhibitors utilizing a 2,6-disubstituted isonicotinic scaffold A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Division of Pharmaceutical Sciences of the James L Winkle College of Pharmacy by Philip T. Cherian M. Pharm. University of Mumbai June 2001 Committee Chair: James J. Knittel, Ph.D. Abstract Phosphatidylinositol 3-OH kinases (PI3Ks) are dual specific lipid and protein kinases that catalyze the synthesis of the lipid second messenger Phosphatidylinositol-3,4,5- trisphosphate (PIP3) and influence multiple cellular processes including cell growth, proliferation, survival and motility. PI3Ks are divided into three classes I, II and III and the class I contains four isoforms, namely p110, , and . Of these, the p110 isoform (PI3K) is an important therapeutic target in cancer as the PIK3CA gene that encodes the p110 catalytic subunit is frequently mutated in a variety of cancers. Though several classes of compounds that inhibit the class I enzymes have been reported, development of inhibitors selective for the PI3Kstill remains a major challenge. In accordance with the ongoing research efforts towards the development of isoform selective inhibitors, we explored the differences between the p110 and binding sites using a structure-based approach. The study entailed the building of a p110 homology model, development of a novel scaffold that provided the ease of assembly and diversification and designing of focused chemical libraries based on our modeling studies. Advancement in protein structure prediction methods has simplified the process of obtaining reliable 3D structures of target proteins. Using the p110 protein sequence and X-ray structure of p110, a homology model of p110 was constructed and refined. This model proved to be in good agreement with the later published X-ray structure of p110 (2rd0). Using this model and literature analysis of PI3K inhibitors, we designed the 2,6- disubstituted isonicotinic scaffold for our study. This scaffold was evaluated for its synthetic feasibility and biological activity by designing, synthesizing and testing an initial set of derivatives. These compounds inhibited the activity of the recombinant iii purified PI3Kand in our in vitro lipid kinase assay and showed inhibition of PI3K- dependent survival of cell lines derived from the hematopoietic FL5.12 cells. The most potent compound in the series (compound 28) showed potency in the low micromolar range with 7-fold selectivity for PI3K. The chemistry developed during the synthesis of the above series provided straightforward access to three chemical libraries. Accordingly, the three regions of the scaffold were modified in order to explore the hydrogen bonding, bulk and polarity as predicted by our model. These modifications led to the development of compound 63 which showed >10-fold selectivity for PI3K vs. PI3K and was more potent in the cell assay than previous compounds. Based on our docking studies, the selectivity of this compound can be attributed to its interaction with Arg770 and Trp780 of p110. Overall we demonstrate the utility of homology modeling and the 2,6-disubstituted scaffold for exploring the p110 and binding sites and anticipate that the data generated during this study may be useful toward the development of more potent and selective PI3K and inhibitors. iv Acknowledgements I express my sincere gratitude to my advisor Dr. James J Knittel for his supervision, support and patience throughout the course of my graduate studies and for his contribution towards my professional development. I thank my committee members Dr. David Plas, Dr. Matt Wortman, Dr. Giovanni Pauletti and Dr. Hal Ebetino for their guidance, constructive criticism and encouragement during the course of this project. In addition I would like to thank Dr. David Plas for allowing me to use his facilities for the cell assay, Dr. Matt Wortman for the software and computers for molecular modeling and Dr. Pauletti for facilities for the kinase assay. I thank Jennifer Barger from the Plas Lab for generating and maintaining the cell lines, designing the cell assays and for her tremendous help during the assay. I would also like to thank Dr. Namal Warshakoon for his help with the project. I express my sincere thanks to our lab members Dr. Leonid Koikov and Dr. Eric Hu for their valuable input and advice during the project and otherwise and Dr. Andrew Ruwe for his assistance during my graduate studies. I would like to thank the James L Winkle College of Pharmacy for their support during my graduate studies as well as for the College of Pharmacy Dean’s Pilot project grant that partially funded this project. I thank the Genome Research Institute (GRI) for providing the facility and instruments for the project. I thank my friends Nirmal, Amit, Sujeet, Moin, Arjun, Rishikesh and Purnima Kulkarni, Shiv and Bhuvana Vishwanathan, Todd and Sara Porter, Brian Drohan, Paul Tanaka and Mugove Manjengwa for making my stay in Cincinnati a delightful experience. vi I express my sincere thankfulness to my brother and his family for their love and support. Above all, I express my deepest gratitude to my parents for their ever increasing love, support and encouragement over the years which have made this accomplishment possible. vii Table of Contents Abstract.............................................................................................................................iii Acknowledgements .......................................................................................................... vi Table of contents ............................................................................................................ viii List of illustrations........................................................................................................... xi List of tables ................................................................................................................... xiii List of abbreviations ...................................................................................................... xiv I. Introduction PI3K family......................................................................................................... 4 Therapeutic applications of PI3K/Akt pathway................................................ 16 PI3K inhibitors.................................................................................................. 20 Inhibitors and crystal structures........................................................................ 26 Goal, hypothesis and specific aims................................................................... 30 II. Molecular modeling Protein structure prediction............................................................................... 32 Modeling programs and servers........................................................................ 44 Limitations of structure prediction methods..................................................... 46 Homology modeling in drug design and development..................................... 47 Results and discussion - Constructing the p110 homology model ................................................ 50 - Designing the scaffold .............................................................................. 53 Conclusions....................................................................................................... 58 Limitations of our modeling ............................................................................. 61 viii III. Evaluating the 2,6-disubstituted isonicotinic scaffold Introduction....................................................................................................... 65 Results and discussion - Chemistry.................................................................................................. 69 - Biological Evaluation ............................................................................... 73 Conclusions....................................................................................................... 83 Limitations of our experimental methods......................................................... 83 IV. Structure-activity relationship (SAR) of 2,6-disubstituted isonicotinic derivatives Introduction....................................................................................................... 85 Results and discussion A. Modifications of Carboxylic acid group ...................................................... 87 - Chemistry.................................................................................................. 88 - Biological evaluation................................................................................ 90 B. Substitution of Morpholine .......................................................................... 91 - Chemistry.................................................................................................. 93 - Biological
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