Increasing yield of in vitro transcription reaction with at-line high pressure liquid chromatography monitoring.

The COVID-19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low-throughput, end-point analytics. We implemented a workflow based on rapid at-line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub-3 min read-out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed-batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti-reverse cap analog (ARCA)-capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed-batch approach achieved productivity of 9 mg/ml with 79% capping. The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed-batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.

Why does the Y326I mutant of monoamine oxidase B decompose an endogenous amphetamine at a slower rate than the wild type enzyme? Reaction step elucidated by multiscale molecular simulations.

This work investigates the Y326I point mutation effect on the kinetics of oxidative deamination of phenylethylamine (PEA) catalyzed by the monoamine oxidase B (MAO B) enzyme. PEA is a neuromodulator capable of affecting the plasticity of the brain and is responsible for the mood enhancing effect caused by physical exercise. Due to a similar functionality, PEA is often regarded as an endogenous amphetamine. The rate limiting step of the deamination was simulated at the multiscale level, employing the Empirical Valence Bond approach for the quantum treatment of the involved valence states, whereas the environment (solvated protein) was represented with a classical force field. A comparison of the reaction free energy profiles delivered by simulation of the reaction in the wild type MAO B and its Y326I mutant yields an increase in the barrier by 1.06 kcal mol-1 upon mutation, corresponding to a roughly 6-fold decrease in the reaction rate. This is in excellent agreement with the experimental kinetic studies. Inspection of simulation trajectories reveals possible sources of the point mutation effect, namely vanishing favorable electrostatic interactions between PEA and a Tyr326 side chain and an increased amount of water molecules at the active site due to the replacement of tyrosine by a less spacious isoleucine residue, thereby increasing the dielectric shielding of the catalytic environment provided by the enzyme.

Micro-pharmacokinetics of lidocaine and bupivacaine transfer across a myelinated nerve fiber.

BACKGROUND: The aim of the present study was to predict the time to onset and duration of action of two local anesthetics (lidocaine and bupivacaine) based on experimental dimensions of a typical nerve and experimental octanol/water partition coefficients. METHODS: We began our compilation of experimental data with a numerical solution of the Smoluchowski equation for the transfer of lidocaine and bupivacaine across the axon membrane in the region of the node of Ranvier (axolemma) and across the Schwann cell. The difference between the aqueous and lipid environments of the neuron was simulated by including the coordinate-dependent chemical potential. In the second step, the permeation rates calculated using the diffusion equation were used to solve a system of four ordinary differential equations. This approach allowed us to simulate the cellular environment for a longer time and to compare our model with pharmacokinetic properties (time to onset and duration of action) of local anesthetics from the literature. The behavior of local anesthetics under physiological conditions and in case of local acidosis was also simulated. RESULTS: We demonstrated that local anesthetics cross the axolemma in a time span of less than 1 µs. The time to onset of action, controlled by diffusion from the epineurium to an axon with a typical distance of 500 µm, was 167 s and 186 s for lidocaine and bupivacaine, respectively. The calculated half-life, which is a measure of the duration of action, was 41 min and 328 min for lidocaine and bupivacaine, respectively. CONCLUSIONS: Duration of action is controlled by the storage capacity of lipophilic compartments around the axon, which is higher for bupivacaine but lower in local acidosis. For the latter case, the literature, including textbooks, provides a misinterpretation, namely that protonated species cannot penetrate the membrane.

Brunner syndrome caused by point mutation explained by multiscale simulation of enzyme reaction.

Brunner syndrome is a disorder characterized by intellectual disability and impulsive, aggressive behavior associated with deficient function of the monoamine oxidase A (MAO-A) enzyme. These symptoms (along with particularly high serotonin levels) have been reported in patients with two missense variants in MAO-A (p.R45W and p.E446K). Herein, we report molecular simulations of the rate-limiting step of MAO-A-catalyzed serotonin degradation for these variants. We found that the R45W mutation causes a 6000-fold slowdown of enzymatic function, whereas the E446K mutation causes a 450-fold reduction of serotonin degradation rate, both of which are practically equivalent to a gene knockout. In addition, we thoroughly compared the influence of enzyme electrostatics on the catalytic function of both the wild type MAO-A and the p.R45W variant relative to the wild type enzyme, revealing that the mutation represents a significant electrostatic perturbation that contributes to the barrier increase. Understanding genetic disorders is closely linked to understanding the associated chemical mechanisms, and our research represents a novel attempt to bridge the gap between clinical genetics and the underlying chemical physics.

Local Anesthetics Transfer Across the Membrane: Reproducing Octanol-Water Partition Coefficients by Solvent Reaction Field Methods.

Local anesthetics are one of the most widely used drug classes in clinical practice. Like many other biological molecules, their properties are altered depending on their protonation status, which is dependent on the pH of the environment. We studied the transport energetics of seven local anesthetics from the extracellular fluid across the biological membrane to the axoplasm in order to understand the effect of pH value on their efficacy and other pharmaco-dynamic properties. In this we applied three different methods of solvent reaction field in conjunction with quantum chemical calculations to reproduce experimental values of n-octanol/water partition coefficients for both neutral and protonated forms. Only the SMD method of Cramer and Truhlar was able to reproduce experimental partition coefficient values. The results are discussed in terms of the function of local anesthetics under physiological conditions and in the case of local acidosis.

How Important Is the Use of Cocaine and Amphetamines in the Development of Parkinson Disease? A Computational Study.

We studied dopamine levels in three compartments of the dopaminergic synapse, including the presynaptic neuron cytosol, dopamine storage vesicles, and the synaptic gap. By considering three transport pathways (dopamine transporter (DAT), vesicular transporter (VT), and exocytosis), four simulated scenarios were investigated: homeostasis, application of cocaine, methamphetamine, and reserpine. Recent experiments show that upon cocaine administration, the Drosophila melanogaster DAT permeation rate constant is decreased by 55% and we adopted this value for the human DAT. Amphetamine and methamphetamine block DAT and VT, while reserpine blocks VT; however, their decreased permeation rate constants are not available. A system of three differential equations of dopamine levels as a function of time was developed respectively for the synaptic compartments and was solved numerically. Per computational inference, the cytosol dopamine concentration was noted to increase in the case of methamphetamine and reserpine, but was practically unchanged in the case of the cocaine administration. Accordingly, our study suggests that amphetamines and other substances that block VT, but not cocaine or substances that only block DAT, may be etiologically important in the cytosolic dopamine mediation of neurodegeneration in Parkinson disease/Parkinsonism.