Jiayun Pang

Drug–polymer intermolecular interactions in hot-melt extruded solid dispersions

The purpose of the study was to investigate and identify the interactions within solid dispersions of cationic drugs and anionic polymers processed by hot-melt extrusion (HME) technique. Propranolol HCl (PRP) and diphenhydramine HCl (DPD) were used as model cationic active substances while pH sensitive anionic methacrylic acid based methyl methacrylate copolymers Eudragit L100 (L100) and ethyl acrylate copolymer Eudragit L100-55 (Acryl EZE) (L100-55) were used as polymeric carriers. The extrudates were further characterised using various physicochemical characterisation techniques to determine the morphology, the drug state within the polymer matrices and the type of drug-polymer interactions. Molecular modelling predicted the existence of two possible H-bonding types while the X-ray photon spectroscopy (XPS) advanced surface analysis of the extrudates revealed intermolecular ionic interactions between the API amino functional groups and the polymer carboxylic groups through the formation of hydrogen bonding. The magnitude of the intermolecular interactions varied according to the drug-polymer miscibility.

Evidence To Support the Hypothesis That Promoting Vibrations Enhance the Rate of an Enzyme Catalyzed H-Tunneling Reaction

In recent years there has been a shift away from transition state theory models for H-transfer reactions. Models that incorporate tunneling as the mechanism of H-transfer are now recognized as a better description of such reactions. Central to many models of H-tunneling is the notion that specific vibrational modes of the protein and/or substrate can increase the probability of a H-tunneling reaction, modes that are termed promoting vibrations. Thus far there has been limited evidence that promoting vibrations can increase the rate of H-transfer. In the present communication we examine the single hydride transfer from both NADPH and NADH to FMN in the reductive half-reaction of pentaerythritol tetranitrate reductase (PETNR). We find that there is a significant promoting vibration with NADPH but not with NADH and that the observed rate of hydride transfer is significantly (approximately 15x) faster with NADPH. We rule out differences in rate due to variation in driving force and the donor-acceptor distance, suggesting it is the promoting vibration with NADPH that is the origin of the increased observed rate. This study therefore provides direct evidence that promoting vibrations can lead to an increase in rate.

Barrier Compression Enhances an Enzymatic Hydrogen‐Transfer Reaction

Putting the squeeze on: Hydrostatic pressure causes a shortening of the charge-transfer bond in the binary complex of morphinone reductase and NADH(4) (see diagram). Molecular dynamics simulations suggest that pressure reduces the average reaction barrier width by restricting the conformational space available to the flavin mononucleotide and NADH within the active site. The apparent rate of catalysis increases with pressure.

Parallel pathways and free-energy landscapes for enzymatic hydride transfer probed by hydrostatic pressure

Mutation of an active‐site residue in morphinone reductase leads to a conformationally rich landscape that enhances the rate of hydride transfer from NADH to FMN at standard pressure (1 bar). Increasing the pressure causes interconversion between different conformational substates in the mutant enzyme. While high pressure reduces the donor–acceptor distance in the wild‐type enzyme, increased conformational freedom “dampens” its effect in the mutant.

Molecular dynamics simulation of thermal unfolding of Thermatoga maritima DHFR

Molecular dynamics simulations of the temperature-induced unfolding reaction of native dimeric dihydrofolate reductase from the hyperthermophile Thermatoga maritima (TmDHFR) and the experimentally inaccessible TmDHFR monomer were carried out at 400 K, 450 K and 500 K. The results revealed that the unfolding of TmDHFR subunits followed a similar path to that of the monomeric DHFR from the mesophile E. coli (EcDHFR). An initial collapse of the adenosine-binding domain (ABD) was followed by the loss of the N-terminal and loop domains (NDLD). Interestingly, the elements of the secondary structure of the isolated TmDHFR monomer were maintained for significantly longer periods of time for the hyperthermophilic enzyme, suggesting that subunit stability contributes to the enhanced resistance of TmDHFR to temperature-induced unfolding. The interactions between the subunits of the TmDHFR dimer led to a stabilisation of the NDLD. The hydrogen bonds between residues 140-143 in betaG of one subunit and residues 125-127 in betaF of the other subunit were retained for significant parts of the simulations at all temperatures. These intermolecular hydrogen bonds were lost after the unfolding of the individual subunits. The high stability of the dimer mediated by strong intersubunit contacts together with an intrinsically enhanced stability of the subunits compared to EcDHFR provides a molecular rational for the higher stability of the thermophilic enzyme. The computed unfolding pathways suggest that the partly folded dimer may be a genuine folding intermediate.

Protein motions during catalysis by dihydrofolate reductases.

Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima. We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

Molecular Modeling as a Predictive Tool for the Development of Solid Dispersions

In this study molecular modeling is introduced as a novel approach for the development of pharmaceutical solid dispersions. A computational model based on quantum mechanical (QM) calculations was used to predict the miscibility of various drugs in various polymers by predicting the binding strength between the drug and dimeric form of the polymer. The drug/polymer miscibility was also estimated by using traditional approaches such as Van Krevelen/Hoftyzer and Bagley solubility parameters or Flory-Huggins interaction parameter in comparison to the molecular modeling approach. The molecular modeling studies predicted successfully the drug-polymer binding energies and the preferable site of interaction between the functional groups. The drug-polymer miscibility and the physical state of bulk materials, physical mixtures, and solid dispersions were determined by thermal analysis (DSC/MTDSC) and X-ray diffraction. The produced solid dispersions were analyzed by X-ray photoelectron spectroscopy (XPS), which confirmed not only the exact type of the intermolecular interactions between the drug-polymer functional groups but also the binding strength by estimating the N coefficient values. The findings demonstrate that QM-based molecular modeling is a powerful tool to predict the strength and type of intermolecular interactions in a range of drug/polymeric systems for the development of solid dispersions.

Atomistic insight into the origin of the temperature-dependence of kinetic isotope effects and H-tunnelling in enzyme systems is revealed through combined experimental studies and biomolecular simulation

The physical basis of the catalytic power of enzymes remains contentious despite sustained and intensive research efforts. Knowledge of enzyme catalysis is predominantly descriptive, gained from traditional protein crystallography and solution studies. Our goal is to understand catalysis by developing a complete and quantitative picture of catalytic processes, incorporating dynamic aspects and the role of quantum tunnelling. Embracing ideas that we have spearheaded from our work on quantum mechanical tunnelling effects linked to protein dynamics for H-transfer reactions, we review our recent progress in mapping macroscopic kinetic descriptors to an atomistic understanding of dynamics linked to biological H-tunnelling reactions.

Secondary kinetic isotope effects as probes of environmentally-coupled enzymatic hydrogen tunneling reactions.

The secondary kinetic isotope effect for hydride transfer from NADPH to dihydrofolate catalyzed by dihydrofolate reductase (see traces) is neither temperature dependent nor exalted. In environmentally coupled models of H-tunneling, the secondary isotope effects do not report on promoting motions, but reflect the active site geometry attained immediately prior to H transfer (i.e. the ‘tunnelling ready configuration′).

Assignment of the vibrational spectra of enzyme-bound tryptophan tryptophyl quinones using a combined QM/MM approach

Fourier transform infrared (FTIR) spectroscopy can be used to provide a detailed time-resolved probe of reaction intermediates in enzyme-catalyzed systems. Accurate assignment of the respective chemical species being studied is key to the success of this approach. The plethora of signals from the protein environment, leading to complexity in the spectra, presents a particular challenge. Here we present a combined QM/MM-based approach that can be used to assign key resonances in the FTIR spectrum of tryptophan tryptophyl quinone (TTQ) in the TTQ-dependent quinoprotein aromatic amine dehydrogenase (AADH). We show that consideration of the cofactor alone is not sufficient to identify correctly the experimentally observed resonances-inclusion of the protein is required for this. However, to enable accurate peak assignment, a stepwise approach is needed that builds up increasing levels of complexity from a simple system. This study serves as a benchmark for future QM/MM-based studies to predict the spectroscopic changes during the interconversion of intermediates in the reductive half-reaction catalyzed by AADH, and more generally for using a combined QM/MM approach to calculate spectroscopic data of protein cofactors and cofactor-based adducts.