Covalent bicyclization of protein complexes yields durable quaternary structures.

Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.

A new synergistic model for simulating exercise incorporating control mechanisms at cellular and organ scales.

The physiological response of the cardio-vascular system (CVS) to physical activity is of great importance to those working in sporting research and has profound consequences for the health and well-being of people. Coronary vasodilation and the physiological mechanisms involved in exercise have frequently been the focus of numerical models for simulating exercise. This is partly achieved using the time-varying-elastance (TVE) theory, which prescribes the pressure-volume relationship of the ventricle as a periodic function of time, tuned using empirical data. The empirical foundations of the TVE method however, and its suitability for CVS modelling are frequently questioned. To overcome this challenge, we adopt a different synergistic approach in which a model for the microscale heart muscle (myofibers) activity is embedded within a macro organ-scale CVS model. We developed such a synergistic model by including the coronary flow and various control mechanisms at the circulation level through feedback and feedforward means, and at the microscale (contractile) through the regulation of ATP availability and myofiber force depending on exercise intensity or heart rate. The coronary flow produced by the model displays the well-known 2-phase character of the flow, which is preserved under exercise. The model is tested by simulating reactive hyperemia, which is a transient occlusion of the coronary flow, successfully reproducing the additional coronary flow following the block removal. On-transient exercise results reveal a rise in both cardiac output and mean ventricle pressure as expected. The stroke volume increases initially, but then declines during the latter period of HR rise, corresponding with one of the main physiological responses to exercise. The pressure-volume loop expands during exercise, as systolic pressure rises. The Myocardial oxygen demand increases during exercise and the coronary blood supply increases in response, causing an excess of oxygen supply to the heart. Off-transient exercise recovery is largely a reverse of this response, although the behaviour is slightly more varied, with sudden spikes in coronary resistance. Different levels of fitness and exercise intensity are tested and reveal that the stroke volume rises until a level of myocardial oxygen demand is reached at which point it declines. This level of demand is independent of fitness or exercise intensity. An advantage of our model is demonstrated in the correspondence between the micro and organ scale mechanics so that cellular pathologies can be traced from exercise performance with relatively little computational or experimental expense.

Development of a novel low-order model for atrial function and a study of atrial mechano-electric feedback.

Numerical models of the cardiovascular system have largely focused on the function of the ventricles, with atrial function often neglected. Furthermore, the time-varying elastance method that prescribes the pressure-volume relationship rather than calculating it consistently is frequently used for the ventricles and atrium. This method has yet to be validated however, so its applicability for cardiac modelling is frequently questioned. To overcome this challenge, we propose a synergistic model of left atrium (LA) and left ventricle (LV) by self-consistently integrating various feedback mechanisms among the electro-mechanical and chemical functions of the micro-scale myofiber, the macro-scale dynamics of the LA and LV, the atrioventricular node (AV), and circulation. The model is tested and shown to reproduce the essential features of the atrium cycling, such as the characteristic figure of eight pressure-volume loops. Our model is further developed to investigate the effect of dysfunctions of the mechanical-electric feedback (MEF) in the atrium. Our model not only successfully reproduces key experimental MEF observations such as prolonged action-potential and increases in action-potential magnitude induced by atrial stretch but also shows how MEF and arrhythmia of the atrium lead to a degradation of cardiac output and pumping power with significant consequences. In particular, MEF reproduces arrhythmia such as ectopic and erratic cycling, missed heart beats and restricted function.

Donor-Acceptor Dyads and Triads Employing Core-Substituted Naphthalene Diimides: A Synthetic and Spectro (Electrochemical) Study.

Donor-acceptor dyads and triads comprising core-substituted naphthalene diimide (NDI) chromophores and either phenothiazine or phenoxazine donors are described. Synthesis combined with electrochemical and spectroelectrochemical investigations facilitates characterisation of the various redox states of these molecules, confirming the ability to combine arrays of electron donating and accepting moieties into single species that retain the redox properties of these individual moieties.

An Investigation of Left Ventricular Valve Disorders and the Mechano-Electric Feedback Using a Synergistic Lumped Parameter Cardiovascular Numerical Model.

Cardiac diseases and failure make up one of largest contributions to global mortality and significantly detriment the quality of life for millions of others. Disorders in the valves of the left ventricle are a prominent example of heart disease, with prolapse, regurgitation, and stenoses-the three main valve disorders. It is widely known that mitral valve prolapse increases the susceptibility to cardiac arrhythmia. Here, we investigate stenoses and regurgitation of the mitral and aortic valves in the left ventricle using a synergistic low-order numerical model. The model synergy derives from the incorporation of the mechanical, chemical, and electrical elements. As an alternative framework to the time-varying elastance (TVE) method, it allows feedback mechanisms at work in the heart to be considered. The TVE model imposes the ventricular pressure-volume relationship using a periodic function rather than calculating it consistently. Using our synergistic approach, the effects of valve disorders on the mechano-electric-feedback (MEF) are investigated. The MEF is the influence of cellular mechanics on the electrical activity, and significantly contributes to the generation of arrhythmia. We further investigate stenoses and regurgitation of the mitral and aortic valves and their relationship with the MEF and generation of arrhythmia. Mitral valve stenosis is found to increase the sensitivity to arrhythmia-stimulating systolic stretch, and reduces the sensitivity to diastolic stretch. Aortic valve stenosis does not change the sensitivity to arrhythmia-stimulating stretch, and regurgitation reduces it. A key result is found when valve regurgitation is accompanied by diastolic stretch. In the presence of MEF disorder, ectopic beats become far more frequent when accompanied by valve regurgitation. Therefore, arrhythmia resulting from a disorder in the MEF will be more severe when valve regurgitation is present.

Of problems and opportunities-How to treat and how to not treat crystallographic fragment screening data.

In their recent commentary in Protein Science, Jaskolski et al. analyzed three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, they claimed that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.

Experiences From Developing Software for Large X-Ray Crystallography-Driven Protein-Ligand Studies.

The throughput of macromolecular X-ray crystallography experiments has surged over the last decade. This remarkable gain in efficiency has been facilitated by increases in the availability of high-intensity X-ray beams, (ultra)fast detectors and high degrees of automation. These developments have in turn spurred the development of several dedicated centers for crystal-based fragment screening which enable the preparation and collection of hundreds of single-crystal diffraction datasets per day. Crystal structures of target proteins in complex with small-molecule ligands are of immense importance for structure-based drug design (SBDD) and their rapid turnover is a prerequisite for accelerated development cycles. While the experimental part of the process is well defined and has by now been established at several synchrotron sites, it is noticeable that software and algorithmic aspects have received far less attention, as well as the implications of new methodologies on established paradigms for structure determination, analysis, and visualization. We will review three key areas of development of large-scale protein-ligand studies. First, we will look into new software developments for batch data processing, followed by a discussion of the methodological changes in the analysis, modeling, refinement and deposition of structures for SBDD, and the changes in mindset that these new methods require, both on the side of depositors and users of macromolecular models. Finally, we will highlight key new developments for the presentation and analysis of the collections of structures that these experiments produce, and provide an outlook for future developments.

Mechanically interlocked molecular handcuffs.

The field of mechanically interlocked molecules that employ a handcuff component are reviewed. The variety of rotaxane and catenane structures that use the handcuff motif to interlock different components are discussed and a new nomenclature, distilling diverse terminologies to a single approach, is proposed. By unifying the interpretation of this class of molecules we identify new opportunities for employing this structural unit for new architectures.

Mechanistic insights into the C55-P targeting lipopeptide antibiotics revealed by structure-activity studies and high-resolution crystal structures.

The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C55-P). In this study we describe the design and synthesis of new C55-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C55-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C55-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin.

Selective photoinduced charge separation in perylenediimide-pillar[5]arene rotaxanes.

The ability to control photoinduced charge transfer within molecules represents a major challenge requiring precise control of the relative positioning and orientation of donor and acceptor groups. Here we show that such photoinduced charge transfer processes within homo- and hetero-rotaxanes can be controlled through organisation of the components of the mechanically interlocked molecules, introducing alternative pathways for electron donation. Specifically, studies of two rotaxanes are described: a homo[3]rotaxane, built from a perylenediimide diimidazolium rod that threads two pillar[5]arene macrocycles, and a hetero[4]rotaxane in which an additional bis(1,5-naphtho)-38-crown-10 (BN38C10) macrocycle encircles the central perylenediimide. The two rotaxanes are characterised by a combination of techniques including electron diffraction crystallography in the case of the hetero[4]rotaxane. Cyclic voltammetry, spectroelectrochemistry, and EPR spectroscopy are employed to establish the behaviour of the redox states of both rotaxanes and these data are used to inform photophysical studies using time-resolved infra-red (TRIR) and transient absorption (TA) spectroscopies. The latter studies illustrate the formation of a symmetry-breaking charge-separated state in the case of the homo[3]rotaxane in which charge transfer between the pillar[5]arene and perylenediimide is observed involving only one of the two macrocyclic components. In the case of the hetero[4]rotaxane charge separation is observed involving only the BN38C10 macrocycle and the perylenediimide leaving the pillar[5]arene components unperturbed.

Improving sampling of crystallographic disorder in ensemble refinement.

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallographic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation.

A method for intuitively extracting macromolecular dynamics from structural disorder.

Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an analytical approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.

Crystallographic approach to fragment-based hit discovery against Schistosoma mansoni purine nucleoside phosphorylase.

Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.

Modelling the cardiac response to a mechanical stimulation using a low-order model of the heart.

Heart diseases are one of the leading causes of death worldwide, and a dysfunction of the cardiac electrical mechanisms is responsible for a significant portion of these deaths. One of these mechanisms, the mechano-electric feedback (MEF), is the electrical response of the heart to local mechanical changes in the environment. This electrical response, in turn, leads to macroscopic changes in heart function. In this paper, we demonstrate that the MEF plays a crucial role in mechanical generation and recovery from arrhythmia which has been observed in experimental studies. To this end, we investigate the cardiac response to a mechanical stimulation using a minimal, multiscale model of the heart which couples the organ level dynamics (left ventricular pressure and volume) and contractile dynamics. By including a mechanical stimulation into the model as a (short, sudden) impulse in the muscle microscale stress, we investigate how the timing, amplitude and duration of the impulse affect the cardiac cycle. In particular, when introduced in the diastolic period of the cardiac cycle, the pulse rate can be stabilised, and ectopic beats and bifurcation can be eliminated, either temporarily or permanently. The stimulation amplitude is a key indicator to this response. We find an optimal value of the impulse amplitude above or below which the impulse maximises the stabilisation. As a result a dysfunction of the MEF can be helped using a mechanical stimulation, by allowing the heart to recover its pumping power. On the other hand, when the mechanical stimulation is introduced towards the end of systole, arrhythmia can be generated.

Achieving Efficient Fragment Screening at XChem Facility at Diamond Light Source.

In fragment-based drug discovery, hundreds or often thousands of compounds smaller than ~300 Da are tested against the protein of interest to identify chemical entities that can be developed into potent drug candidates. Since the compounds are small, interactions are weak, and the screening method must therefore be highly sensitive; moreover, structural information tends to be crucial for elaborating these hits into lead-like compounds. Therefore, protein crystallography has always been a gold-standard technique, yet historically too challenging to find widespread use as a primary screen. Initial XChem experiments were demonstrated in 2014 and then trialed with academic and industrial collaborators to validate the process. Since then, a large research effort and significant beamtime have streamlined sample preparation, developed a fragment library with rapid follow-up possibilities, automated and improved the capability of I04-1 beamline for unattended data collection, and implemented new tools for data management, analysis and hit identification. XChem is now a facility for large-scale crystallographic fragment screening, supporting the entire crystals-to-deposition process, and accessible to academic and industrial users worldwide. The peer-reviewed academic user program has been actively developed since 2016, to accommodate projects from as broad a scientific scope as possible, including well-validated as well as exploratory projects. Academic access is allocated through biannual calls for peer-reviewed proposals, and proprietary work is arranged by Diamond's Industrial Liaison group. This workflow has already been routinely applied to over a hundred targets from diverse therapeutic areas, and effectively identifies weak binders (1%-30% hit rate), which both serve as high-quality starting points for compound design and provide extensive structural information on binding sites. The resilience of the process was demonstrated by continued screening of SARS-CoV-2 targets during the COVID-19 pandemic, including a 3-week turn-around for the main protease.

Intranasal powder live attenuated influenza vaccine is thermostable, immunogenic, and protective against homologous challenge in ferrets.

Influenza viruses cause annual seasonal epidemics and sporadic pandemics; vaccination is the most effective countermeasure. Intranasal live attenuated influenza vaccines (LAIVs) are needle-free, mimic the natural route of infection, and elicit robust immunity. However, some LAIVs require reconstitution and cold-chain requirements restrict storage and distribution of all influenza vaccines. We generated a dry-powder, thermostable LAIV (T-LAIV) using Preservation by Vaporization technology and assessed the stability, immunogenicity, and efficacy of T-LAIV alone or combined with delta inulin adjuvant (Advax™) in ferrets. Stability assays demonstrated minimal loss of T-LAIV titer when stored at 25 °C for 1 year. Vaccination of ferrets with T-LAIV alone or with delta inulin adjuvant elicited mucosal antibody and robust serum HI responses in ferrets, and was protective against homologous challenge. These results suggest that the Preservation by Vaporization-generated dry-powder vaccines could be distributed without refrigeration and administered without reconstitution or injection. Given these significant advantages for vaccine distribution and delivery, further research is warranted.

Bicyclic ß-Sheet Mimetics that Target the Transcriptional Coactivator ß-Catenin and Inhibit Wnt Signaling.

Protein complexes are defined by the three-dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein-protein interactions (PPIs). Even though ß-sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure-based design of ß-sheet mimetics targeting the intracellular protein ß-catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of ß-catenin, a macrocyclic peptide was designed and its crystal structure in complex with ß-catenin obtained. Using this structure, we designed a library of bicyclic ß-sheet mimetics employing a late-stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to ß-catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other ß-sheet-mediated PPIs.

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F.

Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

Synthesis and matched molecular pair analysis of covalent reversible inhibitors of the cysteine protease CPB.

Cysteine protease B (CPB) can be targeted by reversible covalent inhibitors that could serve as antileishmanial compounds. Here, sixteen dipeptidyl nitrile derivatives were synthesized, tested against CPB, and analyzed using matched molecular pairs to determine the effects of stereochemistry and p-phenyl substitution on enzyme inhibition. The compound (S)-2-(((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)amino)-N-(1-cyanocyclopropyl)-3-phenylpropanamide (5) was the most potent CPB inhibitor (pKi = 6.82), which was also selective for human cathepsin B (pKi < 5). The inversion of the stereochemistry from S to R was more detrimental to potency when placed at the P2 position than at P3. The p-Br derivatives were more potent than the p-CH3 and p-OCH3 derivatives, probably due to intermolecular interactions with the S3 subsite.

per-Alkoxy-pillar[5]arenes as Electron Donors: Electrochemical Properties of Dimethoxy-Pillar[5]arene and Its Corresponding Rotaxane.

1,4-dimethoxypillar[5]arene undergoes reversible multielectron oxidations forming stable radical cations, a property retained when incorporated in [2]rotaxanes, suggesting that pillar[5]arenes can be employed as viable, yet unreported, electron donors.