Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ.

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.

Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs.

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins.

Decrypting Integrins by Mixed-Solvent Molecular Dynamics Simulations.

Integrins are a family of α/ß heterodimeric cell surface adhesion receptors which are capable of transmitting signals bidirectionally across membranes. They are known for their therapeutic potential in a wide range of diseases. However, the development of integrin-targeting medications has been impacted by unexpected downstream effects including unwanted agonist-like effects. Allosteric modulation of integrins is a promising approach to potentially overcome these limitations. Applying mixed-solvent molecular dynamics (MD) simulations to integrins, the current study uncovers hitherto unknown allosteric sites within the integrin α I domains of LFA-1 (αLß2; CD11a/CD18), VLA-1 (α1ß1; CD49a/CD29), and Mac-1 (αMß2, CD11b/CD18). We show that these pockets are putatively accessible to small-molecule modulators. The findings reported here may provide opportunities for the design of novel allosteric integrin inhibitors lacking the unwanted agonism observed with earlier as well as current integrin-targeting drugs.

Exploring the Binding Pathway of Novel Nonpeptidomimetic Plasmepsin V Inhibitors.

Predicting the interaction modes and binding affinities of virtual compound libraries is of great interest in drug development. It reduces the cost and time of lead compound identification and selection. Here we apply path-based metadynamics simulations to characterize the binding of potential inhibitors to the Plasmodium falciparum aspartic protease plasmepsin V (plm V), a validated antimalarial drug target that has a highly mobile binding site. The potential plm V binders were identified in a high-throughput virtual screening (HTVS) campaign and were experimentally verified in a fluorescence resonance energy transfer (FRET) assay. Our simulations allowed us to estimate compound binding energies and revealed relevant states along binding/unbinding pathways in atomistic resolution. We believe that the method described allows the prioritization of compounds for synthesis and enables rational structure-based drug design for targets that undergo considerable conformational changes upon inhibitor binding.

Proteostasis of Islet Amyloid Polypeptide: A Molecular Perspective of Risk Factors and Protective Strategies for Type II Diabetes.

The possible link between hIAPP accumulation and ß-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin, and zinc on the mechanism of aggregation of hIAPP. The challenges posed by the transient nature of amyloid oligomers, their structural heterogeneity, and the complex nature of their interaction with lipid membranes have resulted in the development of a wide range of biophysical and chemical approaches to characterize the aggregation process. While the cellular processes and factors activating hIAPP-mediated cytotoxicity are still not clear, it has recently been suggested that its impaired turnover and cellular processing by proteasome and autophagy may contribute significantly toward toxic hIAPP accumulation and, eventually, ß-cell death. Therefore, studies focusing on the restoration of hIAPP proteostasis may represent a promising arena for the design of effective therapies. In this review we discuss the current knowledge of the structures and pathology associated with hIAPP self-assembly and point out the opportunities for therapy that a detailed biochemical, biophysical, and cellular understanding of its aggregation may unveil.

Optimized reaction coordinates for analysis of enhanced sampling.

Atomistic simulations of biological processes offer insights at a high level of spatial and temporal resolution, but accelerated sampling is often required for probing timescales of biologically relevant processes. The resulting data need to be statistically reweighted and condensed in a concise yet faithful manner to facilitate interpretation. Here, we provide evidence that a recently proposed approach for the unsupervised determination of optimized reaction coordinate (RC) can be used for both analysis and reweighting of such data. We first show that for a peptide interconverting between helical and collapsed configurations, the optimal RC permits efficient reconstruction of equilibrium properties from enhanced sampling trajectories. Upon RC-reweighting, kinetic rate constants and free energy profiles are in good agreement with values obtained from equilibrium simulations. In a more challenging test, we apply the method to enhanced sampling simulations of the unbinding of an acetylated lysine-containing tripeptide from the bromodomain of ATAD2. The complexity of this system allows us to investigate the strengths and limitations of these RCs. Overall, the findings presented here underline the potential of the unsupervised determination of reaction coordinates and the synergy with orthogonal analysis methods, such as Markov state models and SAPPHIRE analysis.

Flanking sequence preference modulates de novo DNA methylation in the mouse genome.

Mammalian de novo DNA methyltransferases (DNMT) are responsible for the establishment of cell-type-specific DNA methylation in healthy and diseased tissues. Through genome-wide analysis of de novo methylation activity in murine stem cells we uncover that DNMT3A prefers to methylate CpGs followed by cytosines or thymines, while DNMT3B predominantly methylates CpGs followed by guanines or adenines. These signatures are further observed at non-CpG sites, resembling methylation context observed in specialised cell types, including neurons and oocytes. We further show that these preferences result from structural differences in the catalytic domains of the two de novo DNMTs and are not a consequence of differential recruitment to the genome. Molecular dynamics simulations suggest that, in case of human DNMT3A, the preference is due to favourable polar interactions between the flexible Arg836 side chain and the guanine that base-pairs with the cytosine following the CpG. By exchanging arginine to a lysine, the corresponding side chain in DNMT3B, the sequence preference is reversed, confirming the requirement for arginine at this position. This context-dependent enzymatic activity provides additional insights into the complex regulation of DNA methylation patterns.

Antibody binding modulates the dynamics of the membrane-bound prion protein.

Misfolding of the cellular prion protein (PrPC) is associated with lethal neurodegeneration. PrPC consists of a flexible tail (residues 23-123) and a globular domain (residues 124-231) whose C-terminal end is anchored to the cell membrane. The neurotoxic antibody POM1 and the innocuous antibody POM6 recognize the globular domain. Experimental evidence indicates that POM1 binding to PrPC emulates the influence on PrPC of the misfolded prion protein (PrPSc) while the binding of POM6 has the opposite biological response. Little is known about the potential interactions between flexible tail, globular domain, and the membrane. Here, we used atomistic simulations to investigate how these interactions are modulated by the binding of the Fab fragments of POM1 and POM6 to PrPC and by interstitial sequence truncations to the flexible tail. The simulations show that the binding of the antibodies restricts the range of orientations of the globular domain with respect to the membrane and decreases the distance between tail and membrane. Five of the six sequence truncations influence only marginally this distance and the contact patterns between tail and globular domain. The only exception is a truncation coupled to a charge inversion mutation of four N-terminal residues, which increases the distance of the flexible tail from the membrane. The interactions of the flexible tail and globular domain are modulated differently by the two antibodies.

Dynamics of the Histone Acetyltransferase Lysine-Rich Loop in the Catalytic Core of the CREB-Binding Protein.

The tight control of transcriptional coactivators is a fundamental aspect of gene expression in cells. The regulation of the CREB-binding protein (CBP) and p300 coactivators, two paralog multidomain proteins, involves an autoinhibitory loop (AIL) of the histone acetyltransferase (HAT) domain. There is experimental evidence for the AIL engaging with the HAT binding site, thus interrupting the acetylation of histone tails or other proteins. Both CBP and p300 contain a domain of about 110 residues (called the bromodomain) that recognizes histone tails with one or more acetylated lysine side chains. Here, we investigate by molecular dynamics simulations whether the AIL of CBP (residues 1556-1618) acetylated at the side chain of Lys1595 can bind to the bromodomain. The structural instability and fast unbinding kinetics of the AIL from the bromodomain pocket suggest that the AIL is not a ligand of the bromodomain on the same protein chain. This is further supported by the absence of strong and persistent contacts at the binding interface. Furthermore, the simulations of unbinding show an initial fast detachment of the acetylated lysine and a slower phase necessary for complete AIL dissociation. We provide further evidence for the instability of the AIL intramolecular binding by comparison with a natural ligand, the histone peptide H3K56ac, which shows higher stability in the pocket.

Combined computational and cellular screening identifies synergistic inhibition of SARS-CoV-2 by lenvatinib and remdesivir.

Rapid repurposing of existing drugs as new therapeutics for COVID-19 has been an important strategy in the management of disease severity during the ongoing SARS-CoV-2 pandemic. Here, we used high-throughput docking to screen 6000 compounds within the DrugBank library for their potential to bind and inhibit the SARS-CoV-2 3 CL main protease, a chymotrypsin-like enzyme that is essential for viral replication. For 19 candidate hits, parallel in vitro fluorescence-based protease-inhibition assays and Vero-CCL81 cell-based SARS-CoV-2 replication-inhibition assays were performed. One hit, diclazuril (an investigational anti-protozoal compound), was validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro (IC50 value of 29 µM) and modestly inhibited SARS-CoV-2 replication in Vero-CCL81 cells. Another hit, lenvatinib (approved for use in humans as an anti-cancer treatment), could not be validated as a SARS-CoV-2 3 CL main protease inhibitor in vitro, but serendipitously exhibited a striking functional synergy with the approved nucleoside analogue remdesivir to inhibit SARS-CoV-2 replication, albeit this was specific to Vero-CCL81 cells. Lenvatinib is a broadly-acting host receptor tyrosine kinase (RTK) inhibitor, but the synergistic effect with remdesivir was not observed with other approved RTK inhibitors (such as pazopanib or sunitinib), suggesting that the mechanism-of-action is independent of host RTKs. Furthermore, time-of-addition studies revealed that lenvatinib/remdesivir synergy probably targets SARS-CoV-2 replication subsequent to host-cell entry. Our work shows that combining computational and cellular screening is a means to identify existing drugs with repurposing potential as antiviral compounds. Future studies could be aimed at understanding and optimizing the lenvatinib/remdesivir synergistic mechanism as a therapeutic option.

Simulation Studies of Amyloidogenic Polypeptides and Their Aggregates.

Amyloids, fibrillar assembly of (poly)peptide chains, are associated with neurodegenerative illnesses such as Alzheimer's and Parkinson's diseases, for which there are no cures. The molecular mechanisms of the formation of toxic species are still elusive. Some peptides and proteins can form functional amyloid-like aggregates mainly in bacteria and fungi but also in humans. Little is known on the differences in self-assembly mechanisms of functional and pathogenic (poly)peptides. We review atomistic and coarse-grained simulation studies of amyloid peptides in their monomeric, oligomeric, and fibrillar states. Particular emphasis is given to the challenges one faces to characterize at atomic level of detail the conformational space of disordered (poly)peptides and their aggregation. We discuss the difficulties in comparing simulation results and experimental data, and we propose new simulation studies to shed light on the aggregation processes associated with amyloid diseases.

The 3A6-TCR/superagonist/HLA-DR2a complex shows similar interface and reduced flexibility compared to the complex with self-peptide.

T-cell receptor (TCR) recognition of the myelin basic protein (MBP) peptide presented by major histocompatibility complex (MHC) protein HLA-DR2a, one of the MHC class II alleles associated with multiple sclerosis, is highly variable. Interactions in the trimolecular complex between the TCR of the MBP83-99-specific T cell clone 3A6 with the MBP-peptide/HLA-DR2a (abbreviated TCR/pMHC) lead to substantially different proliferative responses when comparing the wild-type decapeptide MBP90-99 and a superagonist peptide, which differs mainly in the residues that point toward the TCR. Here, we investigate the influence of the peptide sequence on the interface and intrinsic plasticity of the TCR/pMHC trimolecular and pMHC bimolecular complexes by molecular dynamics simulations. The intermolecular contacts at the TCR/pMHC interface are similar for the complexes with the superagonist and the MBP self-peptide. The orientation angle between TCR and pMHC fluctuates less in the complex with the superagonist peptide. Thus, the higher structural stability of the TCR/pMHC tripartite complex with the superagonist peptide, rather than a major difference in binding mode with respect to the self-peptide, seems to be responsible for the stronger proliferative response.

Synthesis, radiolabelling and initial biological characterisation of 18F-labelled xanthine derivatives for PET imaging of Eph receptors.

Eph receptor tyrosine kinases, particularly EphA2 and EphB4, represent promising candidates for molecular imaging due to their essential role in cancer progression and therapy resistance. Xanthine derivatives were identified to be potent Eph receptor inhibitors with IC50 values in the low nanomolar range (1-40 nm). These compounds occupy the hydrophobic pocket of the ATP-binding site in the kinase domain. Based on lead compound 1, we designed two fluorine-18-labelled receptor tyrosine kinase inhibitors ([18F]2/3) as potential tracers for positron emission tomography (PET). Docking into the ATP-binding site allowed us to find the best position for radiolabelling. The replacement of the methyl group at the uracil residue ([18F]3) rather than the methyl group of the phenoxy moiety ([18F]2) by a fluoropropyl group was predicted to preserve the affinity of the lead compound 1. Herein, we point out a synthesis route to [18F]2 and [18F]3 and the respective tosylate precursors as well as a labelling procedure to insert fluorine-18. After radiolabelling, both radiotracers were obtained in approximately 5% radiochemical yield with high radiochemical purity (>98%) and a molar activity of >10 GBq µmol-1. In line with the docking studies, first cell experiments revealed specific, time-dependent binding and uptake of [18F]3 to EphA2 and EphB4-overexpressing A375 human melanoma cells, whereas [18F]2 did not accumulate at these cells. Since both tracers [18F]3 and [18F]2 are stable in rat blood, the novel radiotracers might be suitable for in vivo molecular imaging of Eph receptors with PET.

Structural and Dynamic Insights into Redundant Function of YTHDF Proteins.

Three YTH-domain family proteins (YTHDF1, YTHDF2, and YTHDF3) recognize the N6-methyladenosine (m6A) modification of mRNA in cells. However, the redundancy of their cellular functions has been disputed. We investigate their interactions with m6A-containing RNA using X-ray crystallography and molecular dynamics (MD). The new X-ray structures and MD simulations show that the three proteins share identical interactions with the m6A-containing RNA and have similar intrinsic plasticity, thus evidencing the redundant roles of the three proteins in cellular functions.

An ABSINTH-Based Protocol for Predicting Binding Affinities between Proteins and Small Molecules.

The core task in computational drug discovery is to accurately predict binding free energies in receptor-ligand systems for large libraries of putative binders. Here, the ABSINTH implicit solvent model and force field are extended to describe small, organic molecules and their interactions with proteins. We show that an automatic pipeline based on partitioning arbitrary molecules into substructures corresponding to model compounds with known free energies of solvation can be combined with the CHARMM general force field into a method that is successful at the two important challenges a scoring function faces in virtual screening work flows: it ranks known binders with correlation values rivaling that of comparable state-of-the-art methods and it enriches true binders in a set of decoys. Our protocol introduces innovative modifications to common virtual screening workflows, notably the use of explicit ions as competitors and the integration over multiple protein and ligand species differing in their protonation states. We demonstrate the value of modifications to both the protocol and ABSINTH itself. We conclude by discussing the limitations of high-throughput implicit methods such as the one proposed here.

Assessment of the Fragment Docking Program SEED.

The fragment docking program solvation energy for exhaustive docking (SEED) is evaluated on 15 different protein targets, with a focus on enrichment and the hit rate. It is shown that SEED allows for consistent computational enrichment of fragment libraries, independent of the effective hit rate. Depending on the actual target protein, true positive rates ranging up to 27% are observed at a cutoff value corresponding to the experimental hit rate. The impact of variations in docking protocols and energy filters is discussed in detail. Remaining issues, limitations, and use cases of SEED are also discussed. Our results show that fragment library selection or enhancement for a particular target is likely to benefit from docking with SEED, suggesting that SEED is a useful resource for fragment screening campaigns. A workflow is presented for the use of the program in virtual screening, including filtering and postprocessing to optimize hit rates.

Disulfide bridge formation influences ligand recognition by the ATAD2 bromodomain.

The ATPase family, AAA domain-containing protein 2 (ATAD2) has a C-terminal bromodomain, which functions as a chromatin reader domain recognizing acetylated lysine on the histone tails within the nucleosome. ATAD2 is overexpressed in many cancers and its expression is correlated with poor patient outcomes, making it an attractive therapeutic target and potential biomarker. We solved the crystal structure of the ATAD2 bromodomain and found that it contains a disulfide bridge near the base of the acetyllysine binding pocket (Cys1057-Cys1079). Site-directed mutagenesis revealed that removal of a free C-terminal cysteine (C1101) residue greatly improved the solubility of the ATAD2 bromodomain in vitro. Isothermal titration calorimetry experiments in combination with the Ellman's assay demonstrated that formation of an intramolecular disulfide bridge negatively impacts the ligand binding affinities and alters the thermodynamic parameters of the ATAD2 bromodomain interaction with a histone H4K5ac peptide as well as a small molecule bromodomain ligand. Molecular dynamics simulations indicate that the formation of the disulfide bridge in the ATAD2 bromodomain does not alter the structure of the folded state or flexibility of the acetyllysine binding pocket. However, consideration of this unique structural feature should be taken into account when examining ligand-binding affinity, or in the design of new bromodomain inhibitor compounds that interact with this acetyllysine reader module.

A Reader-Based Assay for m6A Writers and Erasers.

We have developed a homogeneous time-resolved fluorescence (HTRF)-based enzyme assay to measure the catalytic activity of N6-methyladenosine (m6A) methyltransferases and demethylases. The assay detects m6A modifications using the natural m6A-binding proteins (m6A readers). The reaction product or substrate m6A-containing RNA and the m6A reader protein are fluorescently labeled such that their proximity during binding initiates Förster resonance energy transfer (FRET). We show that our HTRF assay can be used for high-throughput screening, which will facilitate the discovery of small-molecule modulators of m6A (de)methylases.

Serotonin uptake is required for Rac1 activation in Kras-induced acinar-to-ductal metaplasia in the pancreas.

Pancreatic ductal adenocarcinoma (PDAC), which is the primary cause of pancreatic cancer mortality, is poorly responsive to currently available interventions. Identifying new targets that drive PDAC formation and progression is critical for developing alternative therapeutic strategies to treat this lethal malignancy. Using genetic and pharmacological approaches, we investigated in vivo and in vitro whether uptake of the monoamine serotonin [5-hydroxytryptamine (5-HT)] is required for PDAC development. We demonstrated that pancreatic acinar cells have the ability to readily take up 5-HT in a transport-mediated manner. 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development. Consistent with the central role played by Rac1 in ADM formation, inhibition of the 5-HT transporter Sert (Slc6a4) with fluoxetine reduced ADM formation both in vitro and in vivo in a cell-autonomous manner. In addition, fluoxetine treatment profoundly compromised the stromal reaction and affected the proliferation and lipid metabolism of malignant PDAC cells. We propose that Sert is a promising therapeutic target to counteract the early event of ADM, with the potential to stall the initiation and progression of pancreatic carcinogenesis. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.