Computational and biochemical screening for the discovery of molecules that stabilize ITPA clinical variants

Project Summary/Abstract
Human inosine triphosphate pyrophosphohydrolase (ITPA) is a protective enzyme that is essential for life. ITPA
hydrolyzes inosine triphosphate (ITP) to the monophosphate form (IMP) to keep the nucleoside triphosphate
from interfering with normal cellular processes and/or incorporation into nucleic acids. The P32T variant of the
enzyme can modulate the results of certain treatments for life-threatening diseases, such as cancer, while the
R178C variant can cause a fatal infantile encephalopathy (DEE 35). Our long-term goal is to develop a drug
that can address this orphan disease (DEE 35) and provide better outcomes for P32T individuals. Experiments
and computer simulations from our previous NIH R15 funding demonstrated that the stability of P32T and R178C
proteins is reduced, catalysis is severely compromised for R178C, and that both mutations disrupted the
structural dynamics of lower lobe helix 2 of the enzyme. These results suggest that a small drug-like molecule
could bind to clinically relevant variants of ITPA to help stabilize the protein. Our specific aims are geared
towards identifying molecules that can reverse these ITPA variants defects. In collaboration with the Molecular
Screening Shared Resource (MSSR) lab at UCLA, we plan to perform molecular docking-based virtual screening
of 300,000 small drug-like molecules of the MSSR library. Our hypothesis is that stabilization of the R178C ITPA
by a library molecule will increase enzyme activity, indicating restored activity. Specific Aim 1 will include in silico
and biochemical chemical library screens. The in silico screening will be performed at Eastern Washington
University (EWU) and the compounds will be ranked based on the binding affinity estimation score. At UCLA, we
will perform high throughput screening (HTS) experiments using a standard biochemical assay to test the
enzymatic activity of R178C ITPA in presence of the top-ranked compounds obtained from the in silico virtual
screening approaches. Data generated from reactions containing library molecules will be statistically evaluated
and those that significantly increase ITPA activity will be considered a “hit” molecule and will be further studied
at EWU using biochemical methods (Specific Aim 2) and molecular dynamic (MD) simulations (Specific Aim
3). The confirmatory biochemistry experiments will include an HPLC-based enzyme assay with wild-type, P32T
and R178C ITPA enzymes. These experiments will provide kinetic parameters and EC50 values, which will help
evaluate each hit molecule and gain insight into how the hit molecule affects enzyme activity. The MD simulations
will confirm the stability of the binding and elucidate the dynamic of the interaction of the hit compounds with the
variants binding site. Undergraduate researchers will take part in every step of the project and participate in
computational and biochemical experiments at EWU and HTS experiments at UCLA. This proposal is innovative
because it explores a new avenue for addressing health issues for patients with ITPA variation. The project is
significant because it will provide a platform for drug discovery to address ITPA defects.

Grant Number: 2R15GM112121-02
NIH Institute/Center: NIH
Principal Investigator: Nicholas Burgis