Wayne State University Wayne State University Dissertations January 2020 Non-Canonical Targets, Reaction Kinetics, And Cellular Potency Of Amino Acid-Linked Platinum(ii) Compounds Bett Kimutai Wayne State University Follow this and additional works at: https://digitalcommons.edu/oa_dissertations Part of the Chemistry Commons Recommended Citation Kimutai, Bett, "Non-Canonical Targets, Reaction Kinetics, And Cellular Potency Of Amino Acid-Linked Platinum(ii) Compounds" (2020). Wayne State University Dissertations.edu/oa_dissertations/2360 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. NON-CANONICAL TARGETS, REACTION KINETICS, AND CELLULAR POTENCY OF AMINO ACID-LINKED PLATINUM(II) COMPOUNDS by BETT KIMUTAI DISSERTATION Submitted to Graduate School of Wayne State University Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2020 MAJOR: Chemistry (Biochemistry) Approved By: _____________________________________ Advisor Date _____________________________________ _____________________________________ _____________________________________ DEDICATION To my parents and wife, Mercy Chepngeno Langat, for their love, encouragement, and support.
ii ACKNOWLEDGEMENTS Foremost, I would like to thank my doctoral advisor, Dr. Christine Chow, for taking me into her research lab. Since I joined, Dr. Chow has provided immense guidance and advice that enabled me to grow in research.
Chow also created an enabling environment and opportunities to explore various career paths in preparation of life after graduate school for which I am grateful. I am thankful to all my dissertation committee members, including Dr. Young-Hoon Ahn, Dr. Rodgers, and Dr.
Weilong Hao for their commitment and advice through my PhD dissertation. I also acknowledge Dr. Louis Romano who had earlier served in my committee until his retirement and grateful to Dr. Ahn for having accepted to join the committee later on.
Rodgers and her lab for the collaborative research projects that we had together for a number of years. I am grateful for the great expertise and contribution of Dr. Rodgers and her former student, Dr. He, toward the collaboration.
The collaboration was very fruitful and enabled us to have publications. Working alongside Dr. He was also a great experience as I learnt a lot from her talent and knowledge. I would also like to thank Dr.
Ahn for allowing me to use a number of his research instruments and lab space. I am also grateful to his former student, Dr. Fidelis Ndombera, for training me to perform mammalian cell experiments. During my time as a graduate student I was surrounded by supportive and friendly colleagues.
I am grateful to my past labmates including Dr. Jun Jiang, Dr. Xun Bao, Dr. Gayani Dedduwa-Mudalige, Dr.
Danielle Dremann, Dr. Hyosuk Seo, Dr. Nisansala Muthunayake, Dr. Supuni Thalalla Gamage, and Dr.
Prabuddha Madubashitha for their advice, expertise, and training on various lab techniques. I am thankful to Dr. Supuni Thalalla Gamage for the fruitful discussions that we had in our collaborative projects and appreciate her talented input in some of the challenging experiments. I am also appreciative of the current Chow lab members including Evan Jones, Rabiul Islam, Alan Mlotkowski, and Deepak Shrestha for being collaborative in our research and providing valuable input from their areas of expertise.
All the past and current labmates exemplified comradery in day to day activities that enabled us to grow in science and iii also to be great friends outside of lab. I would like to thank all the former and current undergraduate students in our lab whom I worked with including Andrew Roberts, Marcel L. Jones, Ken Dada, Nicolas Nunez, and Mateusz Sileski. Each of them was very helpful and provided valuable contributions in various projects.
I am also grateful to all the past and current members of Dr. Ahn and Dr. Bhagwat labs for the fruitful interactions that we had while assisting each other in various lab activities or discussions. I would like to thank the staff at Lumigen instrument center (LIC) for providing me with the training and guidance on how to run various instruments.
I particularly acknowledge Dr. Dennis Anderson, Evan Jones, Dr. Jun Jiang, and Dr. Phil Martin for their expertise and assistance in collecting some of the data.
I am also grateful to Dr. Johnna Birbeck, Dr. Olena Danylyuk, and Dr. Nicholas Peraino for training me on the use of various mass spectrometry techniques.
I am thankful for some of the materials provided generously by a number of labs including Dr. Honn, and Dr. Zhihui Qin from where I acquired mammalian cell lines. I am also grateful for some chemical supplies provided generously by Dr.
Klaus Friedrich from University of Detroit Mercy. I acknowledge WSU-BEST and ReBUILDetroit programs for the career development opportunities that they provided. In winter 2018, I had a semester-long internship to explore a teaching career through these programs. I worked and interacted with exceptional people including Dr.
Jazhara Mayes Otoo and Dr. Jeanne Andreoli from University of Detroit Mercy, and Dr. Andrea Matti from Wayne State University during this teaching internship. They provided such immerse guidance and environment for career development.
I am also grateful to my advisor, Dr. Chow, and Dr. Heidi Kenaga who were an integral part of the BEST program and its opportunities. I would also like to acknowledge the WSU chapter of NOBCChE, its officials, and members for working together towards its mission and goals.
Personally, being part of this incredible team gave me an opportunity to develop leadership skills and other soft career skills especially when I served as its vice-president. iv I am grateful to Wayne State University and the Chemistry department for the opportunity to be part of this institution and the department. I am thankful for the environment and all the resources provided by WSU and the department for me to work on my PhD dissertation. I thank Melissa Rochon for her time and resourcefulness through the entire process.
I would like to thank my parents, Joseph Langat and Florence Langat, their love and support through all the years. I would also like to thank Hellen Jones and my friend Dr. Ezra Mutai at Cornell University who have always been encouraging in my professional and personal life. Lastly, I would like to thank my wife, Mercy Langat, for her love, support and patience.
She has been very encouraging, full of life, and a continued source of inspiration since we met. v TABLE OF CONTENTS DEDICATION………………….iii LIST OF TABLES…………………………………………………………….…………………………x LIST OF FIGURES …………………………………………………………………….xi LIST OF SCHEMES…………………………………………………………………….xv LIST OF ABBREVIATIONS……………………………………………………….…………………xvi CHAPTER 1- INTRODUCTION ………………………………………………….2 Introduction to discovery, mechanism of action, and kinetics of cisPt ………………….1 Discovery of cisPt and its application in cancer therapy…………………………….2 Structural components of cisPt…………………………………………………………4 1.3 Mechanism of action, reaction kinetics, and adduct formation of cisPt…………….3 Introduction to alternative biological targets of cisPt……………………………………………12 1.1 RNA as a target of platinum-based compounds……………………………………12 1.2 Proteins as targets of platinum-based compounds……………………………….4 Cellular resistance and toxicity of cisPt; development of cisPt analogues…………………….1 Toxicity and low selectivity of platinum-based cancer agents…………………….2 Cellular resistance to cisPt…………………………………………………………….3 Development of cisPt analogues for anticancer therapeutics…………………….4 Platinum(IV)-based compounds as antitumor agents………………………………23 1.5 Thesis objectives……………………………………………………………………………………25 CHAPTER 2- AMINO ACID-LINKED PLATINUM(II) COMPOUNDS: SYNTHESIS, CHARACTERIZATION, AND METHODS TO INVESTIGATE THEIR KINETICS, ADDUCTS, AND CELLULAR ACTIVITIES ………………………………………….1 Application of amino acids as ligands of cisplatin analogues………………….2 Formation and characterization of non-canonical AAPt-nucleic acids adducts .3 Kinetics of AAPt with DNA/RNA nucleosides and oligonucleotides……………….4 Impact of AAPt compounds on glycosidic bond stability of adducts……………….5 Potency and accumulation of AAPt compounds in human cancer and normal cells ……………………………………………………………………………………………………35 2.2 Human cell lines and cell culture supplies…………………………………………….1 Synthesis and characterization of AlaPt …………………………………………….2 Synthesis and characterization of OrnPt………………………………………………43 2.3 Synthesis and characterization of ArgPt………………………………………………48 2.4 Preparation of aquated AAPt compounds…………………………………………….5 Preparation of nucleosides and oligonucleotides…………………………………….6 Pseudo-first-order reaction kinetics……………………………………………….7 Fitting data into a kinetic equation…………………………………………………….8 Mass analysis of HPLC isolated AAPt-nucleoside adducts .9 NMR spectroscopy of AAPt-nucleoside adducts…………………………………….10 Mass analysis of HPLC isolated AAPt-oligonucleotide adducts………………….11 Reactions of AAPt with poly(Ado) RNA, ethanol precipitation, and LC -MS characterization of adducts……………………………………………………………………58 2.12 Energy-resolved collision-induced dissociation (ER-CID) experiments and survival yield analyses………………………………………………………………………………….13 MTT cytotoxicity assays in human cell lines .14 Quantification of cellular accumulation of platinum compounds………………….61 CHAPTER 3- CHARACTERIZATION OF NON-CANONICAL ADDUCTS FORMED BY AMINO ACID-LINKED PLATINUM(II) COMPOUNDS …………………………………………………….3 Results and discussion …………………………………………………………………….1 Characterization of AAPt-nucleoside adducts ……………………………………….2 Characterization of AAPt-RNA adducts ……………………………………………….2 Structural isomers of AAPt adducts ………….90 CHAPTER 4- REACTION KINETICS OF AMINO ACID-LINKED PLATINUM(II) COMPOUNDS WITH DNA/RNA NUCLEOSIDES AND OLIGONUCLEOTIDES …………………………………91 4.3 Results and discussion …………………………………………………………………………….1 Kinetics of platination reactions with DNA/RNA purine nucleosides ……………….2 Kinetics of platination reactions with DNA/RNA oligonucleotides ………………….106 CHAPTER 5- IMPACT OF AMINO ACID-LINKED PLATINUM(II) COMPOUNDS ON GLYCOSIDIC BOND STABILITY OF ADDUCTS …………………………………………………108 5.3 Results and discussion………………………………………………………………………….1 Non-canonical AAPt-Ado adduct isomers and their differential fragmentation patterns……………………………………………………………………………………….2 Differential activation of AAPt-Ado isomers towards glycosidic bond cleavage….118 viii CHAPTER 6- POTENCY AND ACCUMULATION OF AMINO ACID-LINKED PLATINUM COMPOUNDS IN HUMAN CANCER AND NORMAL CELLS……………………………………120 6.3 Results and discussion………………………………………………………….1 Potency of AAPt compounds in human cancer and normal cells…………….2 Quantification of platinum abundance in cells………………………………….137 CHAPTER 7- CONCLUSIONS AND FUTURE DIRECTIONS………………………….139 APPENDIX A: CHAPTER 6 SUPPORTING INFORMATION………………………………….…………168 ix LIST OF TABLES Table 1. Clinically approved cancer platinum-based drugs………………………………………. Downfield shifts of protons (in ppm) of AlaPt-Ado adducts relative to Ado protons……76 Table 3.
Downfield shifts of protons (in ppm) of OrnPt-Ado adducts relative to Ado protons…. Downfield shifts of protons (in ppm) of ArgPt-Ado adducts relative to Ado protons…. Pseudo-first-order rate constants…………………………………………………………. Rate constants of cisPt reactions with various DNA/RNA constructs…………………99 Table 4.
Pseudo-first-order rate constants………………………………………………………. IC50 (μM) values of cisPt, carboplatin, and oxaliplatin in human cancer cell lines……123 Table 6. Comparison of potency of AAPt compounds, cisPt, and carboplatin in DU145 cell line………………………………………………………………………………………………………. Comparison of potency of cisPt and ArgPt in normal and cancer cell lines………….
IC50 (μM) values ArgPt in comparison with cisPt………………………………………. Accumulation of platinum compounds in prostate cancer and normal cells…………. ICP-MS analysis of diluted samples of cells treated with platinum -based compounds…………………………………………………………………………………………….134 x LIST OF FIGURES Figure 1. Timeline for discovery, development, and applications of cisPt……………………….
The structures of cisplatin (cisPt) and transplatin with their respective configurations……………………………………………………………………………………………. The structural components of cisPt………………………………………………………. Mode of action of cisPt……………………………………………………………………. Reaction kinetics of cisPt and DNA……………………………………………………….
Potential DNA coordination sites for cisPt………………………………………………. Various types of DNA adducts that are formed by cisPt…………. Structure of cisPt-nucleoside adducts…………………………………………………. Crystal structure of cisPt bound to 50S RNA………………….
Crystal structure cisPt bound to a protein……………………………………………. Nucleotide repair mechanism to remove cisPt adducts……………………………. Antitumor platinum-based compounds………………………………………………. Platinum drugs bound to dGuo residues in the major groove of DNA…………….
Ligands of Pt(IV)-based complexes and their roles…………………………………. Structures of Pt(IV)-based anticancer agents…………………………………………. The structures of cisPt and amino acid-linked platinum(II) compounds, AlaPt, OrnPt, and ArgPt………………………………………………………………………………………………. Possible structures of AAPt compounds……………………………………………….
The structures of amino acid-linked platinum(II) compounds…………………………33 Figure 2. The 1H-NMR spectra of L-alanine and AlaPt……………………………………………39 Figure 2. The 13C-NMR spectra of L-alanine and AlaPt………………. The 13C-NMR time course of AlaPt aquation…………………………………….
The 195Pt-NMR spectrum of potassium tetrachloroplatinate(II) and AlaPt………. ESI mass spectrum of AlaPt…………………………………………………. The 1H-NMR spectra of L-ornithine and OrnPt……………………………. The 13C-NMR specta of L-ornithine and OrnPt…………………………….
The 1H-NMR spectra of OrnPt in D2O and DMSO……………………………. ESI mass spectrum of OrnPt……………………………………………………. Crystal structure of OrnPt…………………………………………………………. The 1H-NMR spectra of L-arginine and ArgPt…………………………………………49 Figure 2.
The 13C-NMR spectra of L-arginine and ArgPt…………………………………. Mass analysis of ArgPt using FT-ICR MS………………………………………. HPLC analysis of 5′-d(TTATT)-3′…………………………………………………. MALDI-MS mass spectrum of 5′-d(TTATT)-3′…………………………………….
HPLC analysis of 5′-UUAUU-3′……………………………………………………. MALDI-MS mass spectrum of the 5′-UUAUU-3′…………………………………. HPLC calibration curves for Ado and Guo…………………………………………. Possible coordination sites for cisPt in DNA………………………………………….
The structures of cisPt and amino acid-linked platinum(II) (AAPt) compounds…. HPLC analysis (C18) of platination products……………………………………. Mass spectra of the AlaPt-AdoN1/N3 and AlaPt-AdoN7 fractions………………………. Wide range mass spectra of the AlaPt-Ado fractions………………………………….
Mass spectra of the OrnPt-AdoN1/N3 and OrnPt-AdoN7 fractions………………………72 Figure 3. Wide range mass spectra of the OrnPt-Ado fractions…………………………………72 Figure 3. Mass spectra of the ArgPt-AdoN1/N3 and ArgPt-AdoN7 fractions………………………73 Figure 3. Wide range mass spectra of the ArgPt-Ado fractions…………………………………74 Figure 3.
Mass spectrum of ArgPt-Adomix fraction………………………………………………. The 2D heteronuclear single quantum correlation (HSQC) of OrnPt-AdoN7 and OrnPt- AdoN1/N3 …………………………………………………………………………………………………. Aromatic region of 1H-NMR spectra of AlaPt-Ado HPLC fractions. Aromatic region of 1H-NMR spectra of OrnPt-Ado HPLC fractions.