Abstract
In the development of antiviral drugs that target viral RNA-dependent RNA polymerases, off-target toxicity caused by the inhibition of the human mitochondrial RNA polymerase (POLRMT) is a major liability. Therefore, it is essential that all new ribonucleoside analogue drugs be accurately screened for POLRMT inhibition. A computational tool that can accurately predict NTP binding to POLRMT could assist in evaluating any potential toxicity and in designing possible salvaging strategies. Using the available crystal structure of POLRMT bound to an RNA transcript, here we created a model of POLRMT with an NTP molecule bound in the active site. Furthermore, we implemented a computational screening procedure that determines the relative binding free energy of an NTP analogue to POLRMT by free energy perturbation (FEP), i.e. a simulation in which the natural NTP molecule is slowly transformed into the analogue and back. In each direction, the transformation was performed over 40 ns of simulation on our IBM Blue Gene Q supercomputer. This procedure was validated across a panel of drugs for which experimental dissociation constants were available, showing that NTP relative binding free energies could be predicted to within 0.97 kcal/mol of the experimental values on average. These results demonstrate for the first time that free-energy simulation can be a useful tool for predicting binding affinities of NTP analogues to a polymerase. We expect that our model, together with similar models of viral polymerases, will be very useful in the screening and future design of NTP inhibitors of viral polymerases that have no mitochondrial toxicity.
Highlights
In the development of antiviral drugs that target viral RNAdependent RNA polymerases, off-target toxicity caused by the inhibition of the human mitochondrial RNA polymerase (POLRMT) is a major liability
Binding free energies of NTP analogues to polymerases are hard to compute as averages over molecular dynamics simulations because of the high negative charge of Ϫ4e on nucleoside triphosphates; that is, the response in the fluctuation of the environment to fluctuations in the positions of the charged phosphates and metal ions may be expected to occur on a slower time scale than for systems with less charge polarization, leading to slow convergence of computed binding free energies
POLRMT is a 1230-amino acid protein consisting of three domains, namely the N-terminal extension (NTE) located at the very N terminus of the protein, the N-terminal domain that follows the NTE in the primary sequence, and the C-terminal polymerase domain [1]
Summary
The detailed molecular structure of POLRMT as a closedproduct elongation complex bound to template DNA and a small RNA transcript has been provided by X-ray crystallography, allowing for insights into NTP binding within the active site (Protein Data Bank code 4BOC) [8]. Binding free energies of NTP analogues to polymerases are hard to compute as averages over molecular dynamics simulations because of the high negative charge of Ϫ4e on nucleoside triphosphates; that is, the response in the fluctuation of the environment to fluctuations in the positions of the charged phosphates and metal ions may be expected to occur on a slower time scale than for systems with less charge polarization, leading to slow convergence of computed binding free energies. We note that in FEP, only relative binding free energies are calculated, i.e. binding free-energy differences between different NTPs, instead of absolute binding free energies This facilitates convergence because fluctuations in energy caused by fluctuations in charge density cancel out when the energetic difference is calculated for two NTPs. Relative binding free energies are determined using a thermodynamic cycle, i.e. from the difference between the free energy change for the transformation of the natural NTP to its analogue in aqueous solution and the corresponding free energy change within the solvated protein-bound environment [19]. When combined with experimental testing, it suggests an efficient strategic means for identifying nucleoside antivirals with high specificity and affinity for viral polymerases, leading to the design of safe and effective drugs
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