Reem Al Olaby

Abstract

Hepatitis C Virus (HCV) infects 170 million individuals worldwide. Although several newly FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating all HCV genotypes and subtypes to be used in an interferon-free regimen. On the other hand, malaria is another public health burden that causes 219 million clinical episodes, and 660,000 deaths per year. In addition, 3.3 billion people live in areas at risk of malaria transmission in 106 countries. It is alarming that 86% of deaths caused by malaria globally were in children. Several challenges are faced when treating malaria, such as resistance against drugs that are used in treatment. This necessitates the development of new classes of drugs to overcome resistance. CD81 is a target protein that plays an essential role in the internalization of HCV into hepatocytes. Thus it was also targeted to identify sets of small molecule ligands predicted to bind to several sites that were identified to be involved in HCV infection. Thirty-six ligands predicted by AutoDock to bind to these sites were tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 23 out of 36 of the ligands bound in vitro to the recombinant CD81-LEL protein. In an effort to create new drugs that block hepatitis C virus entry into hepatocytes, we have designed and synthesized a small molecule that targets the HCV E2 glycoprotein binding site on CD81. A selective high affinity ligand (SHAL) (11) was created by linking together two small molecules that were predicted by docking and were shown by experimental methods to bind to the same site on CD81 where E2 binds. SH7153 was found to bind to recombinant CD81-LEL with a Kd of 21 µM but wasn’t found to inhibit HCV infection when tested using Raji cells (antibody neutralizing assays) and HCV infection inhibition assays. This led to the conclusion that the linkers’ lengths should be optimized so as to have a SHAL that fits properly in the desired binding sites. The HCV glycoprotein E2 has also been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. Recently, 2 research groups were able to resolve the core structure of HCV E2 which will largely help providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. By targeting conserved E2 residues among different genotypes and subtypes in the CD81 binding site on HCV E2, one might also be able to develop drugs that block HCV infection in a genotype-independent manner. Using the E2c structure as a template, we have used homology modeling methods to develop a structural model of the E2 protein core (residues 421-645) that includes the three amino acid segments that are not present in the E2c structure. Blind docking to this model was then performed using a library of ~4000 small molecules and a set of 40 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction were selected for experimental testing. Surface Plasmon resonance was used to screen the ligands for binding to recombinant E2 protein and the best binders were subsequently tested to identify compounds that inhibit the infection of hepatocytes by HCV. One compound, 281816, inhibited infection by HCV genotypes 1a, 1b, 2a, 2b, 4a and 6a with IC50’s ranging from 2.2 uM to 4.6 uM. Such inhibitors may represent a new paradigm for HCV treatment. In an attempt to make 281816 more promising, a SHAL prototype was designed using an analogue of 281816 (SH2216). It would be tempting to test the SHAL inhibitory effect and compare it to the 281816’s inhibitory effect. To date, human CD81 (hCD81) is the only human surface protein known to play a role in the process by which sporozoites of several Plasmodium species infect human hepatocytes. Blocking a human receptor that is exploited for the entry process of pathogens has been proven to be a good strategy for fighting drug-resistant mutants. Hence, we targeted the 21 amino acid stretch on CD81 large extracellular loop that was found to be involved in Plasmosium yoleii invasion via virtual screening runs, preliminary binding assays and sporozoite invasion assays. This led to the identification of 4 drug leads that range between moderate and strong inhibitors of infection by Plasmodium yoleii and Plamodium falciparum. Additionally one ligand was found to potentiate the invasion of Plasmodium yoleii.

Department

Biotechnology Program

Graduation Date

6-1-2014

Submission Date

August 2015

First Advisor

Azzazy, Hassan ME

Committee Member 1

Balhorn, Rod

Committee Member 2

Chromy, Brett

Extent

193 p.

Document Type

Doctoral Dissertation

Library of Congress Subject Heading 1

Malaria.

Library of Congress Subject Heading 2

Hepatitis C virus.

Rights

The author retains all rights with regard to copyright. The author certifies that written permission from the owner(s) of third-party copyrighted matter included in the thesis, dissertation, paper, or record of study has been obtained. The author further certifies that IRB approval has been obtained for this thesis, or that IRB approval is not necessary for this thesis. Insofar as this thesis, dissertation, paper, or record of study is an educational record as defined in the Family Educational Rights and Privacy Act (FERPA) (20 USC 1232g), the author has granted consent to disclosure of it to anyone who requests a copy.

Institutional Review Board (IRB) Approval

Not necessary for this item

Comments

My PhD was sponsored by Yousif Jameel Fellowship

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