Enhanced Sampling Molecular Dynamics Simulations Research Articles

On the Binding Mode and Molecular Mechanism of Enzymatic Polyethylene Terephthalate Degradation

Enzymatic degradation of polyethylene terephthlate (PET) by polyester hydrolases is currently subject to intensive research, as it is considered as a potential eco-friendly recycling method for plastic waste. However, the substrate-binding mode and the molecular mechanism of enzymatic PET hydrolysis are still under intense investigation, and controversial hypotheses have been presented. To help unravel the inherent mechanism of biocatalytic PET degradation at the atomic level, we performed solid-state NMR measurements of a cutinase from Thermobifida fusca (TfCut2) embedded in trehalose glasses together with chemically synthesized, amorphous 13C(═O)-labeled oligomeric PET. The resulting ternary enzyme-PET-trehalose glassy system enabled advanced solid-state NMR methods for real-time tracking of the enzymatic PET degradation and the investigation of PET chain dynamics. Combined with enhanced-sampling molecular dynamics simulations, specific enzyme–substrate interactions during the degradation process could also be monitored. Our results demonstrate that the PET chain is first cleaved by TfCut2 in blocks of at least one repeat unit and further to terephthalic acid and ethylene glycol. Moreover, the second step (formation of final hydrolysis products) appears to be rate-limiting in such reactions. The observed dynamic changes and interfacial protein contacts of 13C-labeled PET carbonyl groups suggest that only one PET repeat unit is bound to the enzyme during the degradation process while the rest of the PET chain is only loosely confined to the active site. These results, not accessible by using conventional solution enzyme samples and small nonhydrolyzable substrates, provide a better understanding of the biocatalytic PET degradation mechanism of polyester hydrolases.

Self-assembling peptide biomaterials: Insights from spontaneous and enhanced sampling molecular dynamics simulations

Peptide self-assembly is the process by which peptide molecules aggregate into low dimensional (1D, 2D) or 3D ordered materials with potential applications ranging from drug delivery to electronics. Short peptides are particularly good candidates for forming supramolecular assemblies due to the relatively simple structure and ease of modulating their self-assembly process to achieve required material properties. The experimental resolution of fibrous peptide-based nanomaterials as 3D atomic coordinates remains challenging. For surface-mediated peptide assembly in particular, it is typically not feasible to resolve multiple conformationally distinct surface bound peptide structures by experiment. The mechanisms of peptide self-assembly also remain elusive due to the interchange of complex interactions and multiple time and length scales involved in the self-assembly process. Peptide self-assembly in solution, or mediated by surfaces, is driven by specific interactions between the peptides and water, competing interactions within the peptide and/or between peptide aggregate units and, in the latter case, an interplay of the interactions between peptides and solvent molecules for adsorption onto a proximal surface. Computational methodologies have proven beneficial in elucidating the structures formed during peptide self-assembly and the molecular mechanisms driving it, and hence have scope in facilitating the development of functional peptide-based nanomaterials for medical or biotechnological applications. In this perspective, computational methods that have provided molecular insights into the mechanisms of formation of peptide biomaterials, and the all-atom-resolved structures of peptide assemblies are presented. Established and recently emerged molecular simulation approaches are reviewed with a focus on applications relevant to peptide assembly, including all-atom and coarse-grained “brute force” molecular dynamics methods as well as the enhanced sampling methodologies: umbrella sampling, steered and replica exchange molecular dynamics, and variants of metadynamics. These approaches have been shown to contribute all-atom details not yet available experimentally, to advance our understanding of peptide self-assembly processes and biomaterial formation. The scope of this review includes a summary of the current state of the computational methods, in terms of their strengths and limitations for application to self-assembling peptide biomaterials.

Substituted Oligosaccharides as Protein Mimics: Deep Learning Free Energy Landscapes.

Protein-protein complexes power the majority of cellular processes. Interfering with the formation of such complexes using well-designed mimics is a difficult, yet actively pursued, research endeavor. Due to the limited availability of results on the conformational preferences of oligosaccharides compared to polypeptides, the former have been much less explored than the latter as protein mimics, despite interesting ADMET characteristics. In this work, the conformational landscapes of a series of 956 substituted glucopyranose oligomers of lengths 3 to 12 designed as protein interface mimics are revealed using microsecond-time-scale, enhanced-sampling molecular dynamics simulations. Deep convolutional networks are trained on these large conformational ensembles, to predict the stability of longer oligosaccharide structures from those of their constituent trimer motifs. Deep generative adversarial networks are then designed to suggest plausible conformations for oligosaccharide mimics of arbitrary length and substituent sequences that can subsequently be used as input to docking simulations. Analyzing the performance of the neural networks also yields insights into the intricate collective effects that dominate oligosaccharide conformational dynamics.

Amide-to-ester substitution as a stable alternative to N-methylation for increasing membrane permeability in cyclic peptides

Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides.

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase.

β-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of β-lactamases among pathogenic bacteria limits the efficacy of β-lactam antibiotics. This has created a major public health crisis. The use of β-lactamase inhibitors has proven useful in restoring the activity of β-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-β-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the β-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-β7 and β12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between α3-β7 and β12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.

Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structure-function relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin motor core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the prepowerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.

Stabilized SARS-CoV-2 spike S2 immunogens designed using insights from enhanced sampling molecular dynamics simulations.

The SARS-CoV-2 spike protein is an important molecular machine that facilitates membrane fusion to host cells and is the major antigenic component of vaccines. Notably, full-length prefusion-stabilized spike proteins produce a strong neutralizing antibody response against S1, which contains the N-terminal domain (NTD) and receptor binding domain (RBD). Unfortunately, emerging variants of concern contain numerous mutations, insertions, and deletions within S1 that impact the binding of neutralizing antibodies which may diminish the efficacy of current vaccines. In contrast, S2 is the most conserved region of the spike and is capable of eliciting antibodies that bind broadly to diverse β-coronavirus spikes. Although S2-based immunogens may have potential benefits, a challenge to its usage as an alternative vaccine strategy is the general instability of S2 in the absence of S1. Using enhanced sampling molecular dynamics simulations, we observed that the S2 head adopts an open conformation where individual protomers separate from one another at the apex. Extensive contact map analysis reveals that the formation and breakage of contacts between three helices mediates these opening motions. Through introducing mutations in these helices, we designed a stabilized S2 construct that maintains a closed conformation and has enhanced thermal stability and protein expression. These studies represent the first simulations characterizing S2 conformational dynamics and demonstrate the potential of using enhanced sampling molecular dynamics simulations in the design of stabilized SARS-CoV-2 S2 immunogens.

Voltage-dependent transitions between resting and activated states of the four Cav1.1 voltage sensors observed in MD simulation.

The sliding helix hypothesis describes how, upon depolarization and repolarization of the membrane, the positive gating charges of the S4 helices of voltage-sensing domains (VSDs) one by one move across the membrane electrical field and thereby effect the opening and closing of ion channels. Voltage-gated calcium channels contain four VSDs, which together regulate gating of a common pore. Although these four VSDs are highly homologous, they differ from each other, e.g. in the number of gating charges and in the number and position of their countercharges. Recent atomic-resolution structures of voltage-gated calcium and sodium channels delineate the endpoints of the VSD movement upon depolarization, showing the S4 helix in the activated up-state. While the first structures of VSDs in resting states have been solved for homotetrameric (prokaryotic) channels, resting state structures of the (pseudo-)heterotetrameric eukaryotic sodium and calcium channels are still lacking. Accordingly, structural models showing the transitions from the resting to the activated state and back are still elusive. Here we addressed this problem using enhanced sampling molecular dynamics simulations of the CaV1.1 calcium channel exposed to a strong membrane potential. Indeed, starting from the known up-state, the S4 helices of the four VSDs moved down to positions closely matching the experimentally determined resting state of the bacterial NaVAb channel. Importantly, the trajectories, the transiently formed ionic interactions between gating- and countercharges, and the speed of the S4 transitions differed substantially between the four VSDs. This is consistent with data from recent mutagenesis and voltage-clamp fluorometry studies on CaV1.1 and allows relating the individual VSD movements with their putative roles in the regulation of distinct channel gating properties, as well as their role in the activation of excitation-contraction coupling in skeletal muscle.

PIP2 binding at the voltage sensor domain facilitates KCNQ1 VSD activation and gating.

The cardiac KCNQ1 channel is involved in cell hyperpolarisation, and loss-of-function mutations are associated with Long QT syndrome (LQTS), which can cause sudden cardiac arrest. The KCNQ1 channel is gated by the changes in transmembrane voltage and crucially requires the binding of two phosphatidylinositol-4,5-bisphosphate (PIP2) molecules to open the channel. The recent structure of KCNQ1 has revealed a PIP2 binding site at the voltage-sensor domain (VSD). In addition, previous modelling and electrophysiological studies have proposed another PIP2 binding site at the pore domain. Using enhanced sampling molecular dynamics simulation, we show that PIP2 binding at the VSD facilitates its activation, whereas PIP2 interaction of the pore facilitates VSD-pore coupling by straightening the S6 helix. Our simulation proposes that PIP2 binding at the VSD acts as a counter charge pausing point during the voltage-dependent activation process, lowering the energy barrier during the S4 translocation. Together, our work highlights the importance of PIP2 at the VSD as an activator to the VSD activation, in addition to our previous knowledge as the key coupler between the VSD and the pore.

MYH7 G256E induced changes in myosin biomechanics influence function of myofibril contractile organelles.

We studied the contractile properties of the MYH7 G256E mutation using engineered human stem cell derived cardiomyocytes (hSC-CMs). When hSC-CMs were cultured to 40-45 days post-differentiation and Western blot analysis demonstrated near 100% β-myosin (MYH7). Single sub-cellular contractile organelles (myofibrils) were mounted in a custom built apparatus between a force transducer and linear motor. Rapid solutions switching to maximal activation solution (pCa 4.0) resulted in a doubling of steady-state force and rate of force development for G256E myofibrils compared with wild type (WT) controls. Upon switching to relaxation solution (pCa 9.0), the slope of the initial linear, slow phase relaxation was significantly slower (∼4-fold) for G256E vs WT myofibrils, suggesting the mutation slows the crossbridge detachment rate in loaded contractions. Crossbridge detachment rate can be reduced by slower ADP release or slower ATP binding to myosin. Analysis using stopped flow kinetics showed the ATP binding rate was slightly, but significantly reduced for G256E vs WT myofibrils. Preliminary data from myofibril mechanical analysis suggests that ADP release is also slower with the G256E mutation. Molecular dynamics (MD) simulations of the rigor and myosin.ATP states showed that G256E led to reorganization of hydrogen bonding networks within the transducer region. Enhanced sampling MD simulations of the rigor to post-rigor (ATP-bound) structural transition indicated G256E myosin surmounts a greater energetic barrier describing the binding of switch 1 to ATP, rotation of the transducer backbone, and opening of the actin binding cleft. This increased barrier is attributed to salt bridges formed by the mutant Glu residue. Combined, our results suggest that G256E leads to hypercontraction and delayed relaxation phenotype via modulation of the transducer structure that alters communication between the nucleotide binding pocket and the actin binding surface during the chemo-mechanical cycle.

Molecular determinants of distinctive opioid receptor subtype affinities.

Despite being targeted by over 30% of the drugs available on the market, G-Protein Coupled Receptors (GPCR) have proven to be notoriously difficult to characterize on an atomistic level. While it is known that helix 6 (TM6) movement is necessary for signal transduction, structural details of activation remain elusive and each receptor is generally studied individually, making comparisons between homologous proteins challenging. Given their homology, any newly discovered drug targeting a specific GPCR may be promiscuous, adding work- and cost-intensive dimensions to the drug discovery process. Some of the GPCRs that seem the most appealing as drug targets are opioid receptors. As opioids are potent pain relievers with widespread use and addictive properties, understanding how drugs bind particular opioid receptor subtypes would allow the design of non-addictive derivatives and thus help alleviate the ongoing opioid crisis - in the United States only, it is estimated that 4 million people use opioids recreationally or are dependent on them, with about half overdose-related deaths being related to synthetic opioids. In this work, we have used enhanced-sampling molecular dynamics simulations to explore the conformational dynamics of different subtypes of opioid receptors and uncovered the molecular determinants of their affinity to various endogenous peptides and synthetic opioids. Using enhanced sampling techniques, we have obtained free energy surfaces (FES) of activation of the μ-, κ-, and δ-subtypes. By comparing the metastable states obtained from the FES, we have pinpointed key structural differences relevant to the activation process. These findings will allow the development of simple and accurate structure-activity relationship, which in turn will guide the design of new, less addictive opioids.

Using tetrapeptide GSSS and β-catenin17-48 to study the effects of phosphoserine on polypeptide conformational ensembles.

Phosphorylation is one of the most common post-translational modifications and is known to have roles in protein transport, localization, function, and stability. Studying both the phosphorylated and nonphosphorylated states of a protein is important as they can be correlated to health and disease states, such as in Alzheimer's and Parkinson's disease. β-catenin is a protein that has two independent functions: cell-cell adhesion and transcriptional regulation, and when there is a change in the balance of these roles, β-catenin acts as an oncogene. It is thought that the phosphorylation state of the unstructured N-terminal region plays an important role in regulating the function and localization of β-catenin. Additionally, mutations at Ser33 and Ser37 phosphorylation sites have been associated with several types of cancer including liver, ovarian, and colorectal cancer. The addition of a large, negatively charged group on the serine sidechain is expected to strongly perturb local electric fields, thus influencing backbone conformational sampling and propensity for hydrogen bonding. As such, dipole-dipole interactions are expected to feature prominently in these systems and are best suited to be investigated with an explicitly polarizable force field. Parameters for phosphoserine were recently developed for the Drude polarizable force field. To validate these parameters and study a biologically relevant system, we performed both unbiased and enhanced-sampling molecular dynamics simulations of the phosphorylated and nonphosphorylated states of model tetrapeptide GSSS and a peptide derived from the N-terminal region of β-catenin, including residues 17-48 (β-catenin17-48). We calculated 3JHNHα-couplings to compare against experimental data and assessed the effect of phosphorylation on secondary structure content, peptide-bond dipole moments, and the electric fields acting around the phosphorylation sites. In doing so, we seek to relate electronic structure with polypeptide conformational dynamics.

A combined ligand and target-based virtual screening strategy to repurpose drugs as putrescine uptake inhibitors with trypanocidal activity.

Chagas disease, also known as American trypanosomiasis, is a neglected tropical disease caused by the protozoa Trypanosoma cruzi, affecting nearly 7million people only in the Americas. Polyamines are essential compounds for parasite growth, survival, and differentiation. However, because trypanosomatids are auxotrophic for polyamines, they must be obtained from the host by specific transporters. In this investigation, an ensemble of QSAR classifiers able to identify polyamine analogs with trypanocidal activity was developed. Then, a multi-template homology model of the dimeric polyamine transporter of T. cruzi, TcPAT12, was created with Rosetta, and then refined by enhanced sampling molecular dynamics simulations. Using representative snapshots extracted from the trajectory, a docking model able to discriminate between active and inactive compounds was developed and validated. Both models were applied in a parallel virtual screening campaign to repurpose known drugs as anti-trypanosomal compounds inhibiting polyamine transport in T. cruzi. Montelukast, Quinestrol, Danazol, and Dutasteride were selected for in vitro testing, and all of them inhibited putrescine uptake in biochemical assays, confirming the predictive ability of the computational models. Furthermore, all the confirmed hits proved to inhibit epimastigote proliferation, and Quinestrol and Danazol were able to inhibit, in the low micromolar range, the viability of trypomastigotes and the intracellular growth of amastigotes.

Non-uniform Self-folding of Helical Poly(fluorenevinylene) Derivatives in the Solid State Leading to Amplified Circular Dichroism and Circularly Polarized Light Emission.

An unprecedented non-uniform self-folding of artificial polymer chains composed of turn moieties and stretched segments is presented through the design of a set of optically active poly(fluorene-2,7-diylethene-1,2-diyl) (poly(fluorenevinylene)) derivatives bearing a neomenthyl group and a pentyl group attached at the 9-position of fluorene backbone at various ratios. The folded structure is formed and stabilized through inter-chain interactions in the solid state, leading to remarkably enhanced chiroptical properties (chirality amplification) in terms of circular dichroism (CD) and circularly polarized light (CPL) emission. This phenomenon is rationalized by experimental and theoretical CD and CPL spectral analyses. The polymer arrangements in the solid state were further assessed through transmission electron microscopic observations combined with enhanced sampling molecular dynamics simulations in the solid state revealing the thin film organizations.

Non‐uniform Self‐folding of Helical Poly(fluorenevinylene) Derivatives in the Solid State Leading to Amplified Circular Dichroism and Circularly Polarized Light Emission

AbstractAn unprecedented non‐uniform self‐folding of artificial polymer chains composed of turn moieties and stretched segments is presented through the design of a set of optically active poly(fluorene‐2,7‐diylethene‐1,2‐diyl) (poly(fluorenevinylene)) derivatives bearing a neomenthyl group and a pentyl group attached at the 9‐position of fluorene backbone at various ratios. The folded structure is formed and stabilized through inter‐chain interactions in the solid state, leading to remarkably enhanced chiroptical properties (chirality amplification) in terms of circular dichroism (CD) and circularly polarized light (CPL) emission. This phenomenon is rationalized by experimental and theoretical CD and CPL spectral analyses. The polymer arrangements in the solid state were further assessed through transmission electron microscopic observations combined with enhanced sampling molecular dynamics simulations in the solid state revealing the thin film organizations.

Modified Protein-Water Interactions in CHARMM36m for Thermodynamics and Kinetics of Proteins in Dilute and Crowded Solutions.

Proper balance between protein-protein and protein-water interactions is vital for atomistic molecular dynamics (MD) simulations of globular proteins as well as intrinsically disordered proteins (IDPs). The overestimation of protein-protein interactions tends to make IDPs more compact than those in experiments. Likewise, multiple proteins in crowded solutions are aggregated with each other too strongly. To optimize the balance, Lennard-Jones (LJ) interactions between protein and water are often increased about 10% (with a scaling parameter, λ = 1.1) from the existing force fields. Here, we explore the optimal scaling parameter of protein-water LJ interactions for CHARMM36m in conjunction with the modified TIP3P water model, by performing enhanced sampling MD simulations of several peptides in dilute solutions and conventional MD simulations of globular proteins in dilute and crowded solutions. In our simulations, 10% increase of protein-water LJ interaction for the CHARMM36m cannot maintain stability of a small helical peptide, (AAQAA)3 in a dilute solution and only a small modification of protein-water LJ interaction up to the 3% increase (λ = 1.03) is allowed. The modified protein-water interactions are applicable to other peptides and globular proteins in dilute solutions without changing thermodynamic properties from the original CHARMM36m. However, it has a great impact on the diffusive properties of proteins in crowded solutions, avoiding the formation of too sticky protein-protein interactions.

Improving All-Atom Force Field to Accurately Describe DNA G-Quadruplex Loops.

The DNA G-quadruplex (GQ) displays structural polymorphisms, and interactions between its loops and flanking sequences critically determine which of the diverse GQ conformers is adopted. All-atom molecular dynamics (MD) simulations of GQs are computationally challenging due to slow folding times and force field (ff) artifacts. In an earlier study, a direct folding simulation of the simplest DNA GQ (TBA15) was first reported using a modified version of the AMBER bsc1 ff (bsc1_vdW ff). Despite this successful folding simulation, it was later found that the bsc1_vdW ff is somewhat limited in terms of describing loop structures of GQs, which is problematic because GQ loop regions play key roles in ligand binding to modulate GQ activities. In this study, we further modified the bsc1_vdW ff to enhance the GQ loop prediction by fine-tuning a limited number of van der Waals (vdW) parameters of the standard AMBER bsc1 ff to improve the GQ loop distribution of a target GQ system (three-layered antiparallel GQ; mHtel21). Test simulations of this newly generated ff (bsc1_vdWL ff) on DNA GQs with diverse topologies (hybrid1, hybrid2, and parallel propeller) revealed that loop structures were predicted more accurately than by the bsc1_vdW ff. We consider that enhanced sampling MD simulation methods in combination with bsc1_vdWL provide useful simulation protocols for resolving outstanding issues of DNA GQ folding and GQ/ligand binding at the all-atom level.

Phase diagram of the TIP4P/Ice water model by enhanced sampling simulations.

We studied the phase diagram for the TIP4P/Ice water model using enhanced sampling molecular dynamics simulations. Our approach is based on the calculation of ice-liquid free energy differences from biased coexistence simulations that reversibly sample the melting and growth of layers of ice. We computed a total of 19 melting points for five different ice polymorphs, which are in excellent agreement with the melting lines obtained from the integration of the Clausius-Clapeyron equation. For proton-ordered and fully proton-disordered ice phases, the results are in very good agreement with previous calculations based on thermodynamic integration. For the partially proton-disordered ice III, we find a large increase in stability that is in line with previous observations using direct coexistence simulations for the TIP4P/2005 model. This issue highlights the robustness of the approach employed here for ice polymorphs with diverse degrees of proton disorder. Our approach is general and can be applied to the calculation of other complex phase diagrams.

Conformational Entropy as a Potential Liability of Computationally Designed Antibodies.

In silico antibody discovery is emerging as a viable alternative to traditional in vivo and in vitro approaches. Many challenges, however, remain open to enabling the properties of designed antibodies to match those produced by the immune system. A major question concerns the structural features of computer-designed complementarity determining regions (CDRs), including the role of conformational entropy in determining the stability and binding affinity of the designed antibodies. To address this problem, we used enhanced-sampling molecular dynamics simulations to compare the free energy landscapes of single-domain antibodies (sdAbs) designed using structure-based (DesAb-HSA-D3) and sequence-based approaches (DesAbO), with that of a nanobody derived from llama immunization (Nb10). Our results indicate that the CDR3 of DesAbO is more conformationally heterogeneous than those of both DesAb-HSA-D3 and Nb10, and the CDR3 of DesAb-HSA-D3 is slightly more dynamic than that of Nb10, which is the original scaffold used for the design of DesAb-HSA-D3. These differences underline the challenges in the rational design of antibodies by revealing the presence of conformational substates likely to have different binding properties and to generate a high entropic cost upon binding.

CLIPSing Melanotan-II to Discover Multiple Functionally Selective hMCR Agonists.

The pleiotropic role played by melanocortin receptors (MCRs) in both physiological and pathological processes has stimulated medicinal chemists to develop synthetic agonists/antagonists with improved potency and selectivity. Here, by deploying the Chemical Linkage of Peptide onto Scaffolds strategy, we replaced the lactam cyclization of melanotan II (MT-II), a potent and unselective agonist of human MCRs (hMCRs), with different xylene-derived thioethers. The newly designed peptides displayed binding affinities toward MCRs ranging from the low nanomolar to the sub-micromolar range, highlighting a correlation between the explored linkers and the affinity toward hMCRs. In contrast to the parent peptide (MT-II), compound 5 displayed a remarkable functional selectivity toward the hMC1R. Enhanced sampling molecular dynamics simulations were found to be instrumental in outlining how the employed cyclization strategy affects the peptides' conformational behavior and, as a consequence, the detected hMC1R affinity. Additionally, a model of the peptide 5/hMC1R complex employing the very recently reported cryogenic electron microscopy receptor structure was provided.