CHARMM at 45: Enhancements in Accessibility, Functionality, and Speed.

Since its inception nearly a half century ago, CHARMM has been playing a central role in computational biochemistry and biophysics. Commensurate with the developments in experimental research and advances in computer hardware, the range of methods and applicability of CHARMM have also grown. This review summarizes major developments that occurred after 2009 when the last review of CHARMM was published. They include the following: new faster simulation engines, accessible user interfaces for convenient workflows, and a vast array of simulation and analysis methods that encompass quantum mechanical, atomistic, and coarse-grained levels, as well as extensive coverage of force fields. In addition to providing the current snapshot of the CHARMM development, this review may serve as a starting point for exploring relevant theories and computational methods for tackling contemporary and emerging problems in biomolecular systems. CHARMM is freely available for academic and nonprofit research at https://academiccharmm.org/program.

Structure-Based Design of CBP/EP300 Degraders: When Cooperativity Overcomes Affinity.

We present the development of dCE-2, a structurally novel PROTAC targeting the CREB-binding protein (CBP) and E1A-associated protein (EP300)-two homologous multidomain enzymes crucial for enhancer-mediated transcription. The design of dCE-2 was based on the crystal structure of an in-house bromodomain (BRD) inhibitor featuring a 3-methyl-cinnoline acetyl-lysine mimic acetyl-lysine mimic discovered by high-throughput fragment docking. Our study shows that, despite its modest binding affinity to CBP/EP300-BRD, dCE-2's remarkable protein degradation activity stems from its good cooperativity, which we demonstrate by the characterization of its ternary complex formation both in vitro and in cellulo. Molecular dynamics simulations indicate that in aqueous solvents, this active degrader populates both folded and extended conformations, which are likely to promote cell permeability and ternary complex formation, respectively.

Hierarchical Protofilament Intertwining Rules the Formation of Mixed-Curvature Amyloid Polymorphs.

Amyloid polymorphism is a hallmark of almost all amyloid species, yet the mechanisms underlying the formation of amyloid polymorphs and their complex architectures remain elusive. Commonly, two main mesoscopic topologies are found in amyloid polymorphs characterized by non-zero Gaussian and mean curvatures: twisted ribbons and helical fibrils, respectively. Here, a rich heterogeneity of configurations is demonstrated on insulin amyloid fibrils, where protofilament packing can occur, besides the common polymorphs, also in a combined mode forming mixed-curvature polymorphs. Through AFM statistical analysis, an extended array of heterogeneous architectures that are rationalized by mesoscopic theoretical arguments are identified. Notably, an unusual fibrillization pathway is also unraveled toward mixed-curvature polymorphs via the widespread recruitment and intertwining of protofilaments and protofibrils. The results present an original view of amyloid polymorphism and advance the fundamental understanding of the fibrillization mechanism from single protofilaments into mature amyloid fibrils.

Structure-Based Design of a Potent and Selective YTHDC1 Ligand.

N6-Adenosine methylation (m6A) is a prevalent post-transcriptional modification of mRNA, with YTHDC1 being the reader protein responsible for recognizing this modification in the cell nucleus. Here, we present a protein structure-based medicinal chemistry campaign that resulted in the YTHDC1 inhibitor 40, which shows an equilibrium dissociation constant (Kd) of 49 nM. The crystal structure of the complex (1.6 Å resolution) validated the design. Compound 40 is selective against the cytoplasmic m6A-RNA readers YTHDF1-3 and YTHDC2 and shows antiproliferative activity against the acute myeloid leukemia (AML) cell lines THP-1, MOLM-13, and NOMO-1. For the series of compounds that culminated into ligand 40, the good correlation between the affinity in the biochemical assay and antiproliferative activity in the THP-1 cell line provides evidence of YTHDC1 target engagement in the cell. The binding to YTHDC1 in the cell is further supported by the cellular thermal shift assay. Thus, ligand 40 is a tool compound for studying the role of YTHDC1 in AML.

Small-Molecule Inhibitors of the m7G-RNA Writer METTL1.

We discovered the first inhibitors of the m7G-RNA writer METTL1 by high-throughput docking and an enzymatic assay based on luminescence. Eleven compounds, which belong to three different chemotypes, show inhibitory activity in the range 40-300 µM. Two adenine derivatives identified by docking have very favorable ligand efficiency of 0.34 and 0.31 kcal/mol per non-hydrogen atom, respectively. Molecular dynamics simulations provide evidence that the inhibitors compete with the binding of the cosubstrate S-adenosyl methionine to METTL1. We also present a soakable crystal form that was used to determine the structure of the complex of METTL1 with sinefungin at a resolution of 1.85 Å.

Proteolysis Targeting Chimera Degraders of the METTL3-14 m6A-RNA Methyltransferase.

Methylation of adenine N6 (m6A) is the most frequent RNA modification. On mRNA, it is catalyzed by the METTL3-14 heterodimer complex, which plays a key role in acute myeloid leukemia (AML) and other types of blood cancers and solid tumors. Here, we disclose the first proteolysis targeting chimeras (PROTACs) for an epitranscriptomics protein. For designing the PROTACs, we made use of the crystal structure of the complex of METTL3-14 with a potent and selective small-molecule inhibitor (called UZH2). The optimization of the linker started from a desfluoro precursor of UZH2 whose synthesis is more efficient than that of UZH2. The first nine PROTAC molecules featured PEG- or alkyl-based linkers, but only the latter showed cell penetration. With this information in hand, we synthesized 26 PROTACs based on UZH2 and alkyl linkers of different lengths and rigidity. The formation of the ternary complex was validated by a FRET-based biochemical assay and an in vitro ubiquitination assay. The PROTACs 14, 20, 22, 24, and 30, featuring different linker types and lengths, showed 50% or higher degradation of METTL3 and/or METTL14 measured by Western blot in MOLM-13 cells. They also showed substantial degradation on three other AML cell lines and prostate cancer cell line PC3.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Disrupting Dimeric ß-Amyloid by Electric Fields.

The early oligomers of the amyloid Aß peptide are implicated in Alzheimer's disease, but their transient nature complicates the characterization of their structure and toxicity. Here, we investigate the stability of the minimal toxic species, i.e., ß-amyloid dimers, in the presence of an oscillating electric field. We first use deep learning (AlphaFold-multimer) for generating initial models of Aß42 dimers. The flexibility and secondary structure content of the models are then analyzed by multiple runs of molecular dynamics (MD). Structurally stable models are similar to ensemble representatives from microsecond-long MD sampling. Finally, we employ the validated model as the starting structure of MD simulations in the presence of an external oscillating electric field and observe a fast decay of ß-sheet content at high field strengths. Control simulations using the helical dimer of the 42-residue leucine zipper peptide show higher structural stability than the Aß42 dimer. The simulation results provide evidence that an external electric field (oscillating at 1 GHz) can disrupt amyloid oligomers which should be further investigated by experiments with brain organoids in vitro and eventually in vivo.

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.

The catalytic mechanism of the RNA methyltransferase METTL3.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on mRNA in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a bisubstrate analogue representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release catalysed by METTL3, and suggests that the latter step is rate-limiting. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.

Reevaluation of bromodomain ligands targeting BAZ2A.

BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2-ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off-targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2-ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC-based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP-238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands.

Structure-Based Design of Inhibitors of the m6A-RNA Writer Enzyme METTL3.

Methyltransferase-like 3 (METTL3) and METTL14 form a heterodimeric complex that catalyzes the most abundant internal mRNA modification, N6-methyladenosine (m6A). METTL3 is the catalytic subunit that binds the co-substrate S-adenosyl methionine (SAM), while METTL14 is involved in mRNA binding. The m6A modification provides post-transcriptional level control over gene expression as it affects almost all stages of the mRNA life cycle, including splicing, nuclear export, translation, and decay. There is increasing evidence for an oncogenic role of METTL3 in acute myeloid leukemia. Here, we use structural and dynamic details of the catalytic subunit METTL3 for developing small-molecule inhibitors that compete with SAM. Starting from a hit identified by high-throughput docking, protein crystallography and molecular dynamics simulations were employed to guide the optimization of inhibitory activity. The potency was successfully improved by 8000-fold as measured by a homogeneous time-resolved fluorescence assay. The optimized compound is selective against the off-targets RNA methyltransferases METTL1 and METTL16.

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.

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.

Rapid ex vivo reverse genetics identifies the essential determinants of prion protein toxicity.

The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208 -H140 hydrogen bond ("H-latch") between the α2-α3 and ß2-α2 loops of PrPC . Importantly, GDL that suppresses the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced 19 individual PrPC variants to PrPC -deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K27 or R27 ) within PrPC . Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-type PrPC . K27 may represent an actionable target for compounds aimed at preventing prion-related neurodegeneration.

Ginkgolic acids inhibit SARS-CoV-2 and its variants by blocking the spike protein/ACE2 interplay.

Targeting the interaction between the spike protein receptor binding domain (S-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and angiotensin-converting enzyme 2 (ACE2) is a potential therapeutic strategy for treating coronavirus disease 2019 (COVID-19). However, we still lack small-molecule drug candidates for this target due to the missing knowledge in the hot spots for the protein-protein interaction. Here, we used NanoBiT technology to identify three Ginkgolic acids from an in-house traditional Chinese medicine (TCM) library, and they interfere with the S-RBD/ACE2 interplay. Our pseudovirus assay showed that one of the compounds, Ginkgolic acid C17:1 (GA171), significantly inhibits the entry of original SARS-CoV-2 and its variants into the ACE2-overexpressed HEK293T cells. We investigated and proposed the binding sites of GA171 on S-RBD by combining molecular docking and molecular dynamics simulations. Site-directed mutagenesis and surface plasmon resonance revealed that GA171 specifically binds to the pocket near R403 and Y505, critical residues of S-RBD for S-RBD interacting with ACE2. Thus, we provide structural insights into developing new small-molecule inhibitors and vaccines against the proposed S-RBD binding site.

Spiking burstiness and working memory in the human medial temporal lobe.

Persistent activity has commonly been considered to be a hallmark of working memory (WM). Recent evidence indicates that neuronal discharges in the medial temporal lobe (MTL) are compatible with WM neural patterns observed in cortical areas. However, the characterization of this activity rarely consists of measurements other than firing rates of single neurons. Moreover, a varied repertoire of firing dynamics has been reported in the MTL regions, which motivate the more detailed examination of the relationships between WM processes and discharge patterns undertaken here. Specifically, we investigate' at different resolution levels, firing irregularities in electrode recordings from the hippocampus, amygdala, and the entorhinal cortex of epileptic patients during a WM task. We show that some types of (ir)regularities predict response times of the patients depending on the trial periods under consideration. Prominent burst activity at the population level is observed in the amygdala and entorhinal cortex during memory retrieval. In general, regular and bursty neurons contribute to the decoding of the memory load, yet they display important differences across the three anatomical areas. Our results suggest that nonrandom (non-Poisson) patterns are relevant for WM, which calls for the development and use of statistics complementary to mere spike counts.

Interactions of Curcumin's Degradation Products with the Aß42 Dimer: A Computational Study.

Amyloid-ß (Aß) dimers are the smallest toxic species along the amyloid-aggregation pathway and among the most populated oligomeric accumulations present in the brain affected by Alzheimer's disease (AD). A proposed therapeutic strategy to avoid the aggregation of Aß into higher-order structures is to develop molecules that inhibit the early stages of aggregation, i.e., dimerization. Under physiological conditions, the Aß dimer is highly dynamic and does not attain a single well-defined structure but is rather characterized by an ensemble of conformations. In a recent study, a highly heterogeneous library of conformers of the Aß dimer was generated by an efficient sampling method with constraints based on ion mobility mass spectrometry data. Here, we make use of the Aß dimer library to study the interaction with two curcumin degradation products, ferulic aldehyde and vanillin, by molecular dynamics (MD) simulations. Ensemble docking and MD simulations are used to provide atomistic detail of the interactions between the curcumin degradation products and the Aß dimer. The simulations show that the aromatic residues of Aß, and in particular 19FF20, interact with ferulic aldehyde and vanillin through π-π stacking. The binding of these small molecules induces significant changes on the 16KLVFF20 region.

Identification of a BAZ2A-Bromodomain Hit Compound by Fragment Growing.

BAZ2A is an epigenetic regulator affecting transcription of ribosomal RNA. It is overexpressed in aggressive and recurrent prostate cancer, promoting cellular migration. Its bromodomain is characterized by a shallow and difficult-to-drug pocket. Here, we describe a structure-based fragment-growing campaign for the identification of ligands of the BAZ2A bromodomain. By combining docking, competition binding assays, and protein crystallography, we have extensively explored the interactions of the ligands with the rim of the binding pocket, and in particular ionic interactions with the side chain of Glu1820, which is unique to BAZ2A. We present 23 high-resolution crystal structures of the holo BAZ2A bromodomain and analyze common bromodomain/ligand motifs and favorable intraligand interactions. Binding of some of the compounds is enantiospecific, with affinity in the low micromolar range. The most potent ligand has an equilibrium dissociation constant of 7 µM and a good selectivity over the paralog BAZ2B bromodomain.

Fragment Ligands of the m6A-RNA Reader YTHDF2.

We report 17 small-molecule ligands that compete with N6-methyladenosine (m6A) for binding to the m6A-reader domain of YTHDF2 (YT521-B homology domain family 2). We determined their binding mode at high resolution by X-ray crystallography and quantified their affinity by a fluorescence-based binding assay. 6-Cyclopropyluracil and a pyrazolopyrimidine derivative have favorable ligand efficiencies of 0.47 and 0.38 kcal mol-1 per non-hydrogen atom, respectively. They represent useful starting points for hit optimization.