Optimizing Excipient Properties to Prevent Aggregation in Biopharmaceutical Formulations.

Excipients are included within protein biotherapeutic solution formulations to improve colloidal and conformational stability but are generally not designed for the specific purpose of preventing aggregation and improving cryoprotection in solution. In this work, we have explored the relationship between the structure and antiaggregation activity of excipients by utilizing coarse-grained molecular dynamics modeling of protein-excipient interaction. We have studied human serum albumin as a model protein, and we report the interaction of 41 excipients (polysorbates, fatty alcohol ethoxylates, fatty acid ethoxylates, phospholipids, glucosides, amino acids, and others) in terms of the reduction of solvent accessible surface area of aggregation-prone regions, proposed as a mechanism of aggregation prevention. Polyoxyethylene sorbitan had the greatest degree of interaction with aggregation-prone regions, decreasing the solvent accessible surface area of APRs by 20.7 nm2 (40.1%). Physicochemical descriptors generated by Mordred are employed to probe the structure-property relationship using partial least-squares regression. A leave-one-out cross-validated model had a root-mean-square error of prediction of 4.1 nm2 and a mean relative error of prediction of 0.077. Generally, longer molecules with a large number of alcohol-terminated PEG units tended to interact more, with qualitatively different protein interactions, wrapping around the protein. Shorter or less ethoxylated compounds tend to form hemimicellar clusters at the protein surface. We propose that an improved design would feature many short chains of 5 to 10 PEG units in many distinct branches and at least some hydrophobic content in the form of medium-length or greater aliphatic chains (i.e., six or more carbon atoms). The combination of molecular dynamics simulation and quantitative modeling is an important first step in an all-purpose protein-independent model for the computer-aided design of stabilizing excipients.

Cysteine-Selective Modification of Peptides and Proteins via Desulfurative C-C Bond Formation.

The site-selective modification of peptides and proteins facilitates the preparation of targeted therapeutic agents and tools to interrogate biochemical pathways. Among the numerous bioconjugation techniques developed to install groups of interest, those that generate C(sp3 )-C(sp3 ) bonds are significantly underrepresented despite affording proteolytically stable, biogenic linkages. Herein, a visible-light-mediated reaction is described that enables the site-selective modification of peptides and proteins via desulfurative C(sp3 )-C(sp3 ) bond formation. The reaction is rapid and high yielding in peptide systems, with comparable translation to proteins. Using this chemistry, a range of moieties is installed into model systems and an effective PTM-mimic is successfully integrated into a recombinantly expressed histone.

AI4Green: An Open-Source ELN for Green and Sustainable Chemistry.

An Electronic Laboratory Notebook (ELN) combining features, including data archival, collaboration tools, and green and sustainability metrics for organic chemistry, is presented. AI4Green is a web-based application, available as open-source code and free to use. It offers the core functionality of an ELN, namely, the ability to store reactions securely and share them among different members of a research team. As users plan their reactions and record them in the ELN, green and sustainable chemistry is encouraged by automatically calculating green metrics and color-coding hazards, solvents, and reaction conditions. The interface links a database constructed from data extracted from PubChem, enabling the automatic collation of information for reactions. The application's design facilitates the development of auxiliary sustainability applications, such as our Solvent Guide. As more reaction data are captured, subsequent work will include providing "intelligent" sustainability suggestions to the user.

Effect of Oriented Electric Fields on Biologically Relevant Iron-Sulfur Clusters: Tuning Redox Reactivity for Catalysis.

Enzyme-based iron-sulfur clusters, exemplified in families such as hydrogenases, nitrogenases, and radical S-adenosylmethionine enzymes, feature in many essential biological processes. The functionality of biological iron-sulfur clusters extends beyond simple electron transfer, relying primarily on the redox activity of the clusters, with a remarkable diversity for different enzymes. The active-site structure and the electrostatic environment in which the cluster resides direct this redox reactivity. Oriented electric fields in enzymatic active sites can be significantly strong, and understanding the extent of their effect on iron-sulfur cluster reactivity can inform first steps toward rationally engineering their reactivity. An extensive systematic density functional theory-based screening approach using OPBE/TZP has afforded a simple electric field-effect representation. The results demonstrate that the orientation of an external electric field of strength 28.8 MV cm-1 at the center of the cluster can have a significant effect on its relative stability in the order of 35 kJ mol-1. This shows clear implications for the reactivity of iron-sulfur clusters in enzymes. The results also demonstrate that the orientation of the electric field can alter the most stable broken-symmetry state, which further has implications on the directionality of initiated electron-transfer reactions. These insights open the path for manipulating the enzymatic redox reactivity of iron-sulfur cluster-containing enzymes by rationally engineering oriented electric fields within the enzymes.

Alchemical Free Energy Methods Applied to Complexes of the First Bromodomain of BRD4.

Accurate and rapid predictions of the binding affinity of a compound to a target are one of the ultimate goals of computer aided drug design. Alchemical approaches to free energy estimations follow the path from an initial state of the system to the final state through alchemical changes of the energy function during a molecular dynamics simulation. Herein, we explore the accuracy and efficiency of two such techniques: relative free energy perturbation (FEP) and multisite lambda dynamics (MSλD). These are applied to a series of inhibitors for the bromodomain-containing protein 4 (BRD4). We demonstrate a procedure for obtaining accurate relative binding free energies using MSλD when dealing with a change in the net charge of the ligand. This resulted in an impressive comparison with experiment, with an average difference of 0.4 ± 0.4 kcal mol-1. In a benchmarking study for the relative FEP calculations, we found that using 20 lambda windows with 0.5 ns of equilibration and 1 ns of data collection for each window gave the optimal compromise between accuracy and speed. Overall, relative FEP and MSλD predicted binding free energies with comparable accuracy, an average of 0.6 kcal mol-1 for each method. However, MSλD makes predictions for a larger molecular space over a much shorter time scale than relative FEP, with MSλD requiring a factor of 18 times less simulation time for the entire molecule space.

Kernel Methods for Predicting Yields of Chemical Reactions.

The use of machine learning methods for the prediction of reaction yield is an emerging area. We demonstrate the applicability of support vector regression (SVR) for predicting reaction yields, using combinatorial data. Molecular descriptors used in regression tasks related to chemical reactivity have often been based on time-consuming, computationally demanding quantum chemical calculations, usually density functional theory. Structure-based descriptors (molecular fingerprints and molecular graphs) are quicker and easier to calculate and are applicable to any molecule. In this study, SVR models built on structure-based descriptors were compared to models built on quantum chemical descriptors. The models were evaluated along the dimension of each reaction component in a set of Buchwald-Hartwig amination reactions. The structure-based SVR models outperformed the quantum chemical SVR models, along the dimension of each reaction component. The applicability of the models was assessed with respect to similarity to training. Prospective predictions of unseen Buchwald-Hartwig reactions are presented for synthetic assessment, to validate the generalizability of the models, with particular interest along the aryl halide dimension.

Vibrational Spectroscopic Map, Vibrational Spectroscopy, and Intermolecular Interaction.

Vibrational spectroscopy is an essential tool in chemical analyses, biological assays, and studies of functional materials. Over the past decade, various coherent nonlinear vibrational spectroscopic techniques have been developed and enabled researchers to study time-correlations of the fluctuating frequencies that are directly related to solute-solvent dynamics, dynamical changes in molecular conformations and local electrostatic environments, chemical and biochemical reactions, protein structural dynamics and functions, characteristic processes of functional materials, and so on. In order to gain incisive and quantitative information on the local electrostatic environment, molecular conformation, protein structure and interprotein contacts, ligand binding kinetics, and electric and optical properties of functional materials, a variety of vibrational probes have been developed and site-specifically incorporated into molecular, biological, and material systems for time-resolved vibrational spectroscopic investigation. However, still, an all-encompassing theory that describes the vibrational solvatochromism, electrochromism, and dynamic fluctuation of vibrational frequencies has not been completely established mainly due to the intrinsic complexity of intermolecular interactions in condensed phases. In particular, the amount of data obtained from the linear and nonlinear vibrational spectroscopic experiments has been rapidly increasing, but the lack of a quantitative method to interpret these measurements has been one major obstacle in broadening the applications of these methods. Among various theoretical models, one of the most successful approaches is a semiempirical model generally referred to as the vibrational spectroscopic map that is based on a rigorous theory of intermolecular interactions. Recently, genetic algorithm, neural network, and machine learning approaches have been applied to the development of vibrational solvatochromism theory. In this review, we provide comprehensive descriptions of the theoretical foundation and various examples showing its extraordinary successes in the interpretations of experimental observations. In addition, a brief introduction to a newly created repository Web site (http://frequencymap.org) for vibrational spectroscopic maps is presented. We anticipate that a combination of the vibrational frequency map approach and state-of-the-art multidimensional vibrational spectroscopy will be one of the most fruitful ways to study the structure and dynamics of chemical, biological, and functional molecular systems in the future.

Structural variation of protein-ligand complexes of the first bromodomain of BRD4.

The bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) family, plays a key role in several diseases, especially cancers. With increased interest in BRD4 as a therapeutic target, many X-ray crystal structures of the protein in complex with small molecule inhibitors are publicly available over the recent decade. In this study, we use this structural information to investigate the conformations of the first bromodomain (BD1) of BRD4. Structural alignment of 297 BRD4-BD1 complexes shows a high level of similarity between the structures of BRD4-BD1, regardless of the bound ligand. We employ WONKA, a tool for detailed analyses of protein binding sites, to compare the active site of over 100 of these crystal structures. The positions of key binding site residues show a high level of conformational similarity, with the exception of Trp81. A focused analysis on the highly conserved water network in the binding site of BRD4-BD1 is performed to identify the positions of these water molecules across the crystal structures. The importance of the water network is illustrated using molecular docking and absolute free energy perturbation simulations. 82% of the ligand poses were better predicted when including water molecules as part of the receptor. Our analysis provides guidance for the design of new BRD4-BD1 inhibitors and the selection of the best structure of BRD4-BD1 to use in structure-based drug design, an important approach for faster and more cost-efficient lead discovery.

Benzene, Toluene, and Monosubstituted Derivatives: Diabatic Nature of the Oscillator Strengths of S1 ← S0 Transitions.

For benzene, toluene, aniline, fluorobenzene, and phenol, even sophisticated treatments of electron correlation, such as MRCI and XMS-CASPT2 calculations, show oscillator strengths typically lower than experiment. Inclusion of a simple pseudo-diabatization approach to perturb the S1 state with approximate vibronic coupling to the S2 state for each molecule results in more accurate oscillator strengths. Their absolute values agree better with experiment for all molecules except aniline. When the coupling between the S1 and S2 states is strong at the S0 geometry, the simple diabatization scheme performs less well with respect to the oscillator strengths relative to the adiabatic values. However, we expect the scheme to be useful in many cases where the coupling is weak to moderate (where the maximum component of the coupling has a magnitude less than 1.5 au). Such calculations give an insight into the effects of vibronic coupling of excited states on UV/vis spectra.

Near-Ultraviolet Circular Dichroism and Two-Dimensional Spectroscopy of Polypeptides.

A fully quantitative theory of the relationship between protein conformation and optical spectroscopy would facilitate deeper insights into biophysical and simulation studies of protein dynamics and folding. In contrast to intense bands in the far-ultraviolet, near-UV bands are much weaker and have been challenging to compute theoretically. We report some advances in the accuracy of calculations in the near-UV, which were realised through the consideration of the vibrational structure of the electronic transitions of aromatic side chains.

Machine-Learning-Enabled Virtual Screening for Inhibitors of Lysine-Specific Histone Demethylase 1.

A machine learning approach has been applied to virtual screening for lysine specific demethylase 1 (LSD1) inhibitors. LSD1 is an important anti-cancer target. Machine learning models to predict activity were constructed using Morgan molecular fingerprints. The dataset, consisting of 931 molecules with LSD1 inhibition activity, was obtained from the ChEMBL database. An evaluation of several candidate algorithms on the main dataset revealed that the support vector regressor gave the best model, with a coefficient of determination (R2) of 0.703. Virtual screening, using this model, identified five predicted potent inhibitors from the ZINC database comprising more than 300,000 molecules. The virtual screening recovered a known inhibitor, RN1, as well as four compounds where activity against LSD1 had not previously been suggested. Thus, we performed a machine-learning-enabled virtual screening of LSD1 inhibitors using only the structural information of the molecules.

A Machine Learning Protocol for Predicting Protein Infrared Spectra.

Infrared (IR) absorption provides important chemical fingerprints of biomolecules. Protein secondary structure determination from IR spectra is tedious since its theoretical interpretation requires repeated expensive quantum-mechanical calculations in a fluctuating environment. Herein we present a novel machine learning protocol that uses a few key structural descriptors to rapidly predict amide I IR spectra of various proteins and agrees well with experiment. Its transferability enabled us to distinguish protein secondary structures, probe atomic structure variations with temperature, and monitor protein folding. This approach offers a cost-effective tool to model the relationship between protein spectra and their biological/chemical properties.

Late-Stage Functionalization by Chan-Lam Amination: Rapid Access to Potent and Selective Integrin Inhibitors.

A late-stage functionalization of the aromatic ring in amino acid derivatives is described. The key step is a copper-catalysed diversification of a boronate ester by amination (Chan-Lam reaction) that can be carried out on a complex ß-aryl-ß-amino acid scaffold. This not only considerably extends the substrate scope of amination partners, but also delivers an array of potent and selective integrin inhibitors as potential treatment agents of idiopathic pulmonary fibrosis (IPF). This versatile chemical strategy, which is amenable to high-throughput-array protocols, allows the installation of pharmaceutically valuable heteroaromatic fragments at a late stage by direct coupling to NH heterocycles, leading to compounds with drug-like attributes. It thus constitutes a useful addition to the medicinal chemist's repertoire.

Molecular Simulation of αvß6 Integrin Inhibitors.

The urgent need for new treatments for the chronic lung disease idiopathic pulmonary fibrosis (IPF) motivates research into antagonists of the RGD binding integrin αvß6, a protein linked to the initiation and progression of the disease. Molecular dynamics (MD) simulations of αvß6 in complex with its natural ligand, pro-TGF-ß1, show the persistence over time of a bidentate Arg-Asp ligand-receptor interaction and a metal chelate interaction between an aspartate on the ligand and an Mg2+ ion in the active site. This is typical of RGD binding ligands. Additional binding site interactions, which are not observed in the static crystal structure, are also identified. We investigate an RGD mimetic, which serves as a framework for a series of potential αvß6 antagonists. The scaffold includes a derivative of the widely utilized 1,8-naphthyridine moiety, for which we present force field parameters, to enable MD and relative free energy perturbation (FEP) simulations. The MD simulations highlight the importance of hydrogen bonding and cation-π interactions. The FEP calculations predict relative binding affinities, within 1.5 kcal mol-1, on average, of experiments.

Möbius and Hückel Cyclacenes with Dewar and Ladenburg Defects.

Cyclacene nanobelts have not been synthesized in over 60 years and remain one of the last unsynthesized building blocks of carbon nanotubes. Recent work has predicted that Hückel-cyclacenes containing Dewar benzenoid ring isomers are the most stable isomeric forms for several of the smaller sizes of cyclacene belts. Here, we give a more complete picture of the isomers that are possible within these nanobelt systems by simulating embedded Ladenburg (prismane) benzenoid rings in Hückel-[n]cyclacenes (n = 5-14) and embedded Dewar benzenoid rings in twisted Möbius-[n]cyclacenes (n = 9-14). The Möbius-[9]cyclacene isomer containing one Dewar benzenoid defect and the Hückel-[5]cyclacene isomer containing two maximally spaced Ladenburg benzenoid defects are found to be more stable than their conventional Kekulé benzenoid ring counterparts. The isomers that contain Dewar and Ladenburg benzenoid rings have larger electronic singlet-triplet energy gaps and lower polyradical character when compared with the conventional isomers.

Computed optical spectra of SARS-CoV-2 proteins.

Treatment for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes Covid-19, may well be predicated on knowledge of the structures of protein of this virus. However, often these cannot be determined easily or quickly. Herein, we provide calculated circular dichroism (CD) spectra in the far- and near-UV, and infra-red (IR) spectra in the amide I region for experimental structures and computational models of SARS-CoV-2 proteins. The near-UV CD spectra offer greatest sensitivity in assessing the accuracy of models.

Quantum chemical calculations of tryptophan → heme electron and excitation energy transfer rates in myoglobin.

The development of optical multidimensional spectroscopic techniques has opened up new possibilities for the study of biological processes. Recently, ultrafast two-dimensional ultraviolet spectroscopy experiments have determined the rates of tryptophan → heme electron transfer and excitation energy transfer for the two tryptophan residues in myoglobin (Consani et al., Science, 2013, 339, 1586). Here, we show that accurate prediction of these rates can be achieved using Marcus theory in conjunction with time-dependent density functional theory. Key intermediate residues between the donor and acceptor are identified, and in particular the residues Val68 and Ile75 play a critical role in calculations of the electron coupling matrix elements. Our calculations demonstrate how small changes in structure can have a large effect on the rates, and show that the different rates of electron transfer are dictated by the distance between the heme and tryptophan residues, while for excitation energy transfer the orientation of the tryptophan residues relative to the heme is important. © 2017 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Computing infrared spectra of proteins using the exciton model.

The ability to compute from first principles the infrared spectrum of a protein in solution phase representing a biological system would provide a useful connection to atomistic models of protein structure and dynamics. Indeed, such calculations are a vital complement to 2DIR experimental measurements, allowing the observed signals to be interpreted in terms of detailed structural and dynamical information. In this article, we have studied nine structurally and spectroscopically well-characterized proteins, representing a range of structural types. We have simulated the equilibrium conformational dynamics in an explicit point charge water model. Using the resulting trajectories based on MD simulations, we have computed the one and two dimensional infrared spectra in the Amide I region, using an exciton approach, in which a local mode basis of carbonyl stretches is considered. The role of solvent in shifting the Amide I band (by 30 to 50 cm-1 ) is clearly evident. Similarly, the conformational dynamics contribute to the broadening of peaks in the spectrum. The inhomogeneous broadening in both the 1D and 2D spectra reflects the significant conformational diversity observed in the simulations. Through the computed 2D cross-peak spectra, we show how different pulse schemes can provide additional information on the coupled vibrations. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.

Probing Polyoxometalate-Protein Interactions Using Molecular Dynamics Simulations.

The molecular interactions between the CeIV -substituted Keggin anion [PW11 O39 Ce(OH2 )4 ]3- (CeK) and hen egg-white lysozyme (HEWL) were investigated by molecular dynamics simulations. The analysis of CeK was compared with the CeIV -substituted Keggin dimer [(PW11 O39 )2 Ce]10- (CeK2 ) and the ZrIV -substituted Lindqvist anion [W5 O18 Zr(OH2 )(OH)]3- (ZrL) to understand how POM features such as shape, size, charge, or type of incorporated metal ion influence the POM⋅⋅⋅protein interactions. Simulations revealed two regions of the protein in which the CeK anion interacts strongly: cationic sites formed by Arg21 and by Arg45 and Arg68. The POMs chiefly interact with the side chains of the positively charged (arginines, lysines) and the polar uncharged residues (tyrosines, serines, aspargines) via electrostatic attraction and hydrogen bonding with the oxygen atoms of the POM framework. The CeK anion shows higher protein affinity than the CeK2 and ZrL anions, because it is less hydrophilic and it has the right size and shape for establishing interactions with several residues simultaneously. The larger, more negatively charged CeK2 anion has a high solvent-accessible surface, which is sub-optimal for the interaction, while the smaller ZrL anion is highly hydrophilic and cannot efficiently interact with several residues simultaneously.