Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting HIV-1 RRE RNA ...

366 Pages·2012·13.55 MB·English

Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting
HIV-1 RRE RNA and Microbials

Jessica E. Wynn

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in
partial fulfillment of the requirements for the degree of

Doctor of Philosophy
In
Chemistry

Webster L. Santos, Chair
Karen J. Brewer
Felicia A. Etzkorn
Tijana Z. Grove
David G.I. Kingston

July 14, 2016
Blacksburg, VA

Keywords: HIV-1 RRE RNA, Branched Peptide Library, Boron-Acridine, Antimicrobial
Peptides, Unnatural Amino Acids

Copyright (2016), Jessica E. Wynn
Functionalizing Branched Peptides with Unnatural Amino Acids Toward Targeting
HIV-1 RRE RNA and Antimicrobials

Jessica E. Wynn

ABSTRACT

The interaction of the protein Rev with Rev Response Element (RRE) RNA is critical to
the HIV-1 life cycle as this complex is required for the export of singly-spliced and unspliced
mRNAs from the nucleus to the cytoplasm. Disruption of this interaction is considered to be a
powerful strategy towards the development of HIV-1 therapeutics. Therefore, we have
developed several branched peptide libraries containing unnatural amino acids to target the high-
affinity binding site of RRE RNA (RRE IIB), with the idea that branching in peptides can
provide multivalent contacts with folded RNA structures and boost binding affinity and
selectivity for the target. Unnatural amino acids were incorporated into the library design to
encourage non-canonical interactions with the RNA and to improve proteolytic stability.
The on-bead high-throughput screening of our first branched peptide library (46,656
sequences) against HIV-1 RRE RNA generated hit peptides with binding affinities in the low
micromolar range. We demonstrated that branching in the peptide is required for efficient
binding and selectivity towards the RNA, and that the peptides bind a large surface area of RRE
IIB. Introduction of boronic acids into branched peptides boosted selectivity of the peptides for
RRE IIB, and proved to be a novel and tunable mode of binding towards RNA. Additionally, we
revealed that these branched peptide boronic acids (BPBAs) were cell permeable and non-toxic.
One BPBA (BPBA3) bound RRE IIB selectively and was able to inhibit HIV-1 replication in
vitro, revealing enzymatic cleavage of the RNA upon binding.
A second generation BPBA library that introduced acridinyl lysine as an intercalator
(4,096 sequences) was screened against RRE IIB. Several hit compounds bound in the low
nanomolar regime, and a significant number of compounds inhibited HIV-1 replication in vitro.
These BPBAs were also found to severely inhibit the microbial growth of bacteria and fungus,
with MICs as low as 1 µg/mL against Staphylococcus aureus, Candida albicans, and
Escherichia coli. These compounds were also found to significantly inhibit biofilm formation
and growth, and were non-hemolytic.
High-throughput screening of a third generation BPBA library containing all unnatural
amino acids (46,656 sequences) revealed several hits that bound RRE IIB RNA in the nanomolar
range. Sequence motifs present in the hit peptides suggested that the location and composition of
amino acids within the branched peptide structure were important for recognizing the RNA
target. In particular, lead compounds 2C5 and 4B3 demonstrated selectivity towards RRE, and
footprinting experiments combined with SHAPE experiments revealed different interactions of
the peptides with the RNA Toxicity assays revealed no impact on cell viability for the majority
of hit sequences tested up to 100 µM, and several compounds also demonstrated inhibition of
HIV-1 replication.

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ACKNOWLEDGEMENTS
I preface this acknowledgements section with the observation that yes, this section is
long, but rightly so. To all those who have read or will read this dissertation (n=10?), it is
important to realize that indeed, no man (or woman) is an island1 – I have not gotten to the end
of this journey without a little help from my friends2 (and professors, and colleagues…) On the
flip side of things, if you are an advisor, a professor, an instructor, or a colleague to a graduate
student, please realize that even small gestures of compassion or acknowledgement are forever
remembered, especially during the long bouts of struggle and self-doubt that lurk within a
developing young scientist. Thank you all so much for everything you have done to allow me to
succeed in my endeavors. Without further ado…
First, I would like to thank my advisor, Dr. Webster L. Santos, for giving me the
opportunity to conduct research in his laboratory. I appreciate the autonomy and freedom you
have allowed me over the years to develop and execute my projects, which has in turn given me
the confidence to pursue what will hopefully be a successful career as an independent research
scientist. Your tireless efforts and work ethic are an inspiration to me. Thank you for teaching
me about synthetic chemistry - your ability to communicate complex concepts and make them
interesting is unparalleled, and is one of the reasons I was so interested in joining your lab. I also
thank you for your input on preparing and giving presentations, which has greatly improved my
ability to communicate science with others. Additionally, I would like to thank you for
providing the financial support that has allowed me to conduct my research, and for encouraging
and supporting me to attend conferences and present my work. Last but not least, I thank you for
forever altering the way I communicate – ‘at the end of the day’, ‘gratifyingly’, ‘indeed’, and
‘taken together’ are permanently etched into my shriveled brain.3
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I would like to thank my committee members for nurturing my growth as a graduate
student and for helping me improve by providing honest evaluations of my performance at each
stage of my graduate career. I would like to acknowledge Dr. Felicia A. Etzkorn for her positive
influence in my life as an example of a strong, successful female scientist in the department. I
am indebted to you as your critique of my literature review has stuck with me and improved my
scientific writing. I would like to thank Dr. Tijana Z. Grove for lending her analytical expertise
when discussing my research, and for always taking the time to mentor me and offer support,
both professionally and personally. You have a way of making every student feel valued and
respected, and I wish you nothing but success in your future. I would like to thank Dr. David
G.I. Kingston for his valuable critiques of my work over the years, especially with my original
research proposal. I am constantly amazed by your humility and accessibility to students, even
though you are such a distinguished scientist. Lastly, I would like to thank Dr. Karen J. Brewer.
She encouraged me from my very first semester as a graduate student, and provided emotional
support when all seemed hopeless and lost. Although she is no longer with us, I carry her in my
heart during my final sprint to the finish line and know that she would be proud.
I would also like to thank several faculty members within the Chemistry department for
their endless support during my tenure in graduate school. A big thank you goes to Dr. Jim
Tanko, for many reasons. First, thank you for always being supportive of my endeavors and for
always making time to meet with me, even though you are insanely busy as the Chair. Also,
thank you for teaching a class that made me appreciate both letters and numbers in an equation –
I truly learned a great deal in your Chemical Kinetics class, including discovering a level of
perseverance within myself that I have made good use of over the years. I also thank you for
forever changing the way I see time – it’s five colon zero zero! I am appreciative of both Dr.
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John Morris and Dr. Paul Deck for serving as great resources for me during my time at Virginia
Tech, and for always making the student a priority. I would like to thank Dr. Paul Carlier and
Dr. Richard Gandour for teaching me a great deal about organic and medicinal chemistry. Both
of you have been incredibly supportive of my growth as a chemist, from engaging in my work
during poster sessions to allowing me to serve on various committees over the years. I want to
thank Dr. Brian Tissue for all of his kind words of encouragement over the years, and for giving
me the opportunity to serve on the Safety Committee.
A thank you also goes out to all of the professors that have encouraged me before I began
my journey in graduate school. Thank you, Dr. Ed Smith, for being the first professor to take a
chance on me when I started at Virginia Tech as an undergraduate. Your genuine love and
concern for students from all walks of life is appreciated and admired. I would like to thank Dr.
Neal and Kay Castagnoli for mentoring me during my undergraduate years as well, and for
sparking my love of organic chemistry. I would like to thank Dr. Tim Fuhrer for introducing me
to chemistry in high school – who knew we would cross paths again many years later in graduate
school?! The world is a small place sometimes. There have also been several teachers outside
the realm of chemistry that I would like to thank for encouraging me to think critically and for
embracing my curious nature: Carolyn Paulette, Catherine Neale, Suzy Bolton, and Dr. Karl
Precoda all come to mind. Thank you for the immeasurable impact you have had on my life.
I also want to thank many people on the administrative and chemistry support staff that
have helped me navigate the waters of paperwork, broken instruments, busted computers,
missing packages, NMR questions, and stress: Emillie Shephard, Angie Miller, Angie Kritak,
Patty Angus, Roberta Gilbert, Wanda Hensley, Kristen Cox, Melba Edwards, Anna Hawthorne,
Tom Bell, Donald Neel, Larry Jackson, Steve Breeding, John Burleson, Tom Wertalik, Sharelle
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Carbaugh, Geno Iannaccone, and Murthy Shanaiah. I would especially like to thank Joli Huynh
for all her help in keeping me on track to graduate, and for providing emotional support during
the ‘impending doom’ period. Also, a special thanks goes out to Karen Iannaccone, an honorary
member of the Santos group, for helping out with scheduling, reimbursements, and for making
my life so much easier. Finally, I would like to thank Claire Santos (my other Santos boss) for
giving me the opportunity to be one of her teaching assistants for many years. Your support and
encouragement, both inside and outside the teaching classroom, have meant a great deal to me.
Also, without you, I am not sure what we would have eaten at group parties – you are a fabulous
hostess.
I would like to thank all of the collaborators I have had the opportunity to work with over
the years: Dr. W. Keith Ray, Dr. Kristi DeCourcy, Dr. David M. Rekosh, Dr. Joseph O.
Falkinham, Dr. Stuart LeGrice, and Dr. Florian Schubot. I also would like to thank Peggy
Brodie for taking the time to teach me how to handle cell lines when I first started, and Myra
Williams for all of her contributions and hard work on the antimicrobial project. A special thanks
goes out to Dr. Mehdi Ashraf, who has been not only a great resource for all HPLC and MS
questions, but a mentor to me as well. I will always be appreciative of your advice.
Of course, I would like to thank all members of the Santos group for their support over
the years: Mithun Raje, Ken Knott, David Bryson, Brandon Thorpe, Joe Calderone, Wenyu
Zhang, Xi Guo, Amanda Nelson, Molly Congdon, Analyn Carreon, Neeraj Patwhardan, Beth
Childress, Cheryl Peck, Emily Morris, Russell Snead, Hao Li, Matt Nguyen, Brett Rastatter,
Srinath Pashikanti, Astha Verma, Ashley Peralta, and Yumin Dai. You all have been a joy to
work with and I will miss you all very much.
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Amanders – I do not have the words to express how thankful I am that you came into my
life. I never imagined when I met you outside of Davidson during the ACS entrance exams that
you would become one of my most cherished friends. Thanks for all of the 3 am science
discussions with a side of burnt coffee, the hysterical laughing, the cry-screaming, and all of the
memories (Can I graduate?4 NO!) You made graduate school bearable and always seemed to
know what to say to get me to laugh in the midst of adversity. Thank you, Wenyu, for being my
partner in crime on the RNA project for many years and for balancing my “Type A” antics. I
will never forget the wonderful time I had at your house with your family on Chinese New
Year’s. Also, thank you to the other members of the RNA group (David, Jason, Ashley, and
Yumin) for all of your hard work, past and present, to make this collaborative effort a success.
Em, thank you for always making me laugh, and for being a good friend and confidant. And for
sharing my love of furry babies! Beth, aka Sassy Snowflake, thank you for always being so
supportive and willing to help me out, no strings attached. Also, thank you for all of your help in
editing my chapters. I will miss your face. Mol, thank you for standing by me as we mucked
our way through graduate school, and for being my cheerleader at the end. I also want to give a
shout out to the 3023 crew (Brandon, Joe, Cheryl, Neeraj, and Russell) for keeping it real and
providing me a nurturing environment in which I could discuss topics ranging from the price of
kidneys on the black market5 to ‘limbo of the postdoc’6 over dueling banjos.7 That was
definitely my happy place.
A special thanks to Hanlie Wessels and Marwa Abdel Latif for being such amazing
friends over the years. I am forever grateful for your encouragement and love. I also want to
thank my peers for providing emotional support during graduate school: Eugene Camerino, Alex
Eaton, Roberto Padilla, David Morris, Elise Naughton, Bryce and Spencer Kidd, Dan
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Schoonover, Terry Price, Jenn Rowe, Rachel Parker, Kristina Roth, Cherie Epley, and the list
goes on. The experience of graduate school binds us all.
A big, heart-felt thank you goes out to Karen Godbey. You have stood by me as my best
friend through thick and thin, and there is no way I could have made it through to the end
without your love and support. I hope you realize how much you mean to me and how important
your friendship is to me. Our villa in Spain is waiting. I want to thank John Young, aka NY
John, for all of those late night conversations involving my decision to leave a well-paying job
and return to school. You were always so supportive and really pushed me to follow my heart. I
will never forget you and the influence you have had on my life. I wish you were still here on
earth, I miss you and I hope I made you proud. I also would like to give a shout out to my
friends who have supported me at one point or another during my graduate career: Maria
Achruk, Lance Smith, Caleb Anderson, Lynn Hagy, Jeff Shelton, Kim Murray, Anne Hiller, and
Molly Brunner. Your love and support means the world to me.
I would like to thank my furry four-legged children, Pappy, Jamie, and Piper, for
showering me with unconditional love and for maintaining my sanity after long days and nights
in graduate school. I did not rescue you; rather, you rescued me. Also, thank you Pappy, for
your attempts to help me write my manuscripts and dissertation.8 They are much appreciated.
Finally, I would like to thank my family for their love and support. Jimmy, thank you for
never giving me the answers to any of my questions growing up. You inadvertently prepared
your little sister for a career involving problem solving, which has served me well in graduate
school. Also, thank you for always reminding me of my terrible life decision to go to graduate
school.9 And no, I have not cured AIDS yet. Thank you, Mom and Dad, for supporting me as I
pursued my Ph.D. Your unwavering belief that education is the root of all possibility and all of
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your sacrifices over the years have allowed me to pursue my dreams. Without you, none of this
would have even been remotely possible. I love you all very much, more than I could ever
express.

References and notes:
1. Donne, J. Meditation XVII. Devotions upon Emergent Occasions, originally published in
1624.

2. Lennon, J.; McCartney, P. “With a Little Help From My Friends.” Sgt. Pepper’s Lonely
Hearts Club Band. The Beatles. 1967.

3. Refer to any of the author’s published manuscripts or chapters presented in this
dissertation.

4. Jenkins, S.; Cadogan, K. “Graduate.” Third Eye Blind. Third Eye Blind. 1997.

5. Prices vary depending on source. It was concluded that most likely a seasoned graduate
or postdoctoral student in the organic chemistry division would not fetch a high price for
their kidneys, but that first- or second-year students might have more success.

6. Philosophical interpretations of Dante’s Inferno as applied to graduate and postdoctoral
students.

7. Smith, A.; Weissberg, E.; Mandell, S. “Dueling Banjos.” Reuben’s Train (B-side). 1973.

8. The author would like to emphasize that she is the sole author of this dissertation and
previously published work. “Attempts to help” included loud meowing and using all
fourteen pounds of body weight to simulteneously pound all keys on the keyboard via a
flopping mechanism.

9. “Home Away From Homer.” The Simpsons, Season 16, Episode 20. FOX. May 15, 2005.

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