Integrating computational and experimental approaches in discovery and validation of MmpL3 pore domain inhibitors for specific labelling of Mycobacterium tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a significant global health threat. Identifying new druggable targets is crucial for the advancement of drug development. Equally critical is the development of precise methods for monitoring Mtb to effectively combat this disease. Addressing these needs, our study pinpointed the pore domain (PD) of MtbMmpL3 as a new binding site for virtual screening, which led to the discovery of the small molecule ZY27. To confirm the binding site and action mode of ZY27, we employed cosolvent molecular dynamics (CMD), steered molecular dynamics (SMD), and long timescale molecular dynamics (MD) simulations of 5 µs. These in silico studies verified that ZY27 binds to the PD of MtbMmpL3. In antimicrobial activity tests, ZY27 exhibited potent anti-Mtb activity and high selectivity among mycobacterial species. Whole-genome sequencing of spontaneous ZY27-resistant Mtb variants, complemented by acid-fast staining experiments, confirmed that ZY27 specifically targets MtbMmpL3. Utilizing the ligand-protein binding data, we designed and synthesized two solvatochromic fluorescent probes, 27FP1 and 27FP2, based on ZY27. Further investigations through flow cytometry and confocal microscopy confirmed that these probes specifically label Mtb cells via the MtbMmpL3 binding mechanism.

Design and Discovery of New Collagen V-Derived FGF2-Blocking Natural Peptides Inhibiting Lung Squamous Cell Carcinoma In Vitro and In Vivo.

Aberrant FGF2/FGFR signaling is implicated in lung squamous cell carcinoma (LSCC), posing treatment challenges due to the lack of targeted therapeutic options. Designing drugs that block FGF2 signaling presents a promising strategy different from traditional kinase inhibitors. We previously reported a ColVα1-derived fragment, HEPV (127AA), that inhibits FGF2-induced angiogenesis. However, its large size may limit therapeutic application. This study combines rational peptide design, molecular dynamics simulations, knowledge-based prediction, and GUV and FRET assays to identify smaller peptides with FGF2-blocking properties. We synthesized two novel peptides, HBS-P1 (45AA) and HBS-P2 (66AA), that retained the heparin-binding site. Both peptides demonstrated anti-LSCC and antiangiogenesis properties in cell viability and microvessel network induction assays. In two LSCC subcutaneous models, HBS-P1, with its affinity for FGF2 and enhanced penetration ability, demonstrated substantial therapeutic potential without apparent toxicities. Our study provides the first evidence supporting the development of collagen V-derived natural peptides as FGF2-blocking agents for LSCC treatment.

Peptide Drug Design Using Alchemical Free Energy Calculation: An Application and Validation on Agonists of Ghrelin Receptor.

With recent large-scale applications and validations, the relative binding free energy (RBFE) calculated using alchemical free energy methods has been proven to be an accurate measure to probe the binding of small-molecule drug candidates. On the other hand, given the flexibility of peptides, it is of great interest to find out whether sufficient sampling could be achieved within the typical time scale of such calculation, and a similar level of accuracy could be reached for peptide drugs. However, the systematic evaluation of such calculations on protein-peptide systems has been less reported. Most reported studies of peptides were restricted to a limited number of data points or lacking experimental support. To demonstrate the applicability of the alchemical free energy method for protein-peptide systems in a typical real-world drug discovery project, we report an application of the thermodynamic integration (TI) method to the RBFE calculation of ghrelin receptor and its peptide agonists. Along with the calculation, the synthesis and in vitro EC50 activity of relamorelin and 17 new peptide derivatives were also reported. A cost-effective criterion to determine the data collection time was proposed for peptides in the TI simulation. The average of three TI repeats yielded a mean absolute error of 0.98 kcal/mol and Pearson's correlation coefficient (R) of 0.77 against the experimental free energy derived from the in vitro EC50 activity, showing good repeatability of the proposed method and a slightly better agreement than the results obtained from the arbitrary time frames up to 20 ns. Although it is limited by having one target and a deduced binding pose, we hope that this study can add some insights into alchemical free energy calculation of protein-peptide systems, providing theoretical assistance to the development of peptide drugs.

Create artilysins from a recombinant library to serve as bactericidal and antibiofilm agents targeting Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a critical pathogen and novel treatments are urgently needed. The out membrane of P. aeruginosa facilitates biofilm formation and antibiotic resistance, and hinders the exogenous application against Gram-negative bacteria of endolysins. Engineered endolysins are investigated for enhancing antimicrobial activity, exemplified by artilysins. Nevertheless, existing research predominantly relies on laborious and time-consuming approaches of individually artilysin identification. This study proposes a novel strategy for expedited artilysin discovery using a recombinant artilysin library comprising proteins derived from 38 antimicrobial peptides and 8 endolysins. In this library, 19 colonies exhibited growth inhibition against P. aeruginosa exceeding 50 %, and three colonies were designated as dutarlysin-1, dutarlysin-2 and dutarlysin-3. Remarkably, dutarlysin-1, dutarlysin-2 and dutarlysin-3 demonstrated rapid and enhanced antibacterial activity, even minimum inhibitory concentration of them killed approximately 4.93 lg units, 6.75 lg units and 5.36 lg units P. aeruginosa, respectively. Dutarlysins were highly refractory to P. aeruginosa resistance development. Furthermore, 2 µmol/L dutarlysin-1 and dutarlysin-3 effectively eradicated over 76 % of the mature biofilm. These dutarlysins exhibited potential broad-spectrum activity against hospital susceptible Gram-negative bacteria. These results supported the effectiveness of this artilysins discovery strategy and suggested dutarlysin-1 and dutarlysin-3 could be promising antimicrobial agents for combating P. aeruginosa.

Transport Parameters for Combustion Species Based on cAMOEBA Polarizable Force Field.

In this study, we report a simplified yet accurate general AMOEBA polarizable force field for combustion-interested molecular species, denoted as Combustion-AMOEBA or cAMOEBA. By eliminating the permanent atomic dipoles and quadrupoles, retaining the explicit polarization and defining the general atom types of each molecule species, including alkanes, alkenes, alkynes, alcohols, peroxides, and aldehydes, a simplified and general cAMOEBA force field was constructed and validated using the benchmark results obtained at the QCISD(T)/CBS level of theory. In this way, the tedious parametrization step for permanent atomic multipoles of each new molecule in the original AMOEBA (Poltype/MP2) force field could be avoided, hence providing the capability of accurate high-throughput calculation for a large number of molecules at lower computational cost. The averaged difference between the calculated transport parameters, σ and ε, for approximately 100 different molecules and four bath gases (He, Ne, Ar, and N2) using cAMOEBA and AMOEBA (Poltype/MP2) are of 0.09% and 1.27%, respectively, showing a good consistence of the general cAMOEBA force field with the original AMOEBA (Poltype/MP2) force field where the multipole force field parameters were obtained from quantum mechanical calculation for each small molecule. Our results also indicated that the Lorentz-Berthelot combination rule was more applicable than Waldman-Hagler for obtaining the molecular Lennard-Jones parameters of pure gases from one bath gas, while the Waldman-Hagler combination rule was better for obtaining such parameters from all four bath gases. The pure gas parameters obtained using cAMOEBA can be applied to develop high quality transport property database for combustion modeling.

Molecular Dynamics Simulation-Driven Focused Virtual Screening and Experimental Validation of Inhibitors for MTDH-SND1 Protein-Protein Interaction.

Protein-protein interactions (PPIs), in general, are attractive yet challenging drug targets. As a typical PPI, MTDH-SND1 interaction has recently been reported to be a promising drug target to malignant breast cancer and other cancer types. However, the lack of well-defined deep pockets on the MTDH-SND1 interface makes it a tough target for rational drug discovery attempts. To address this issue, in this study, a long time-scale molecular dynamics (MD) simulation-driven focused screening strategy was proposed and reported. A total of 12 virtual hits were purchased and tested in SPR assay, yielding 10 SND1 binders with micromolar or less affinities. As an example, compound L5, the second best hit with a KD of 2.64 µM, was further assayed in MDA-MB-231 breast cancer cells, showing an antiproliferation IC50 value of 57 µM in a CCK8 assay with a dampened interruption between MTDH and SND1 proteins detected by immunofluorescence colocalization imaging. As the most potent small molecule inhibitor in the class so far, our preliminary study combining molecular dynamics simulation and in vitro cellular functional evidence indicates L5 could serve as a lead compound for future optimization or pharmacologic studies, and the MD-driven focused screening strategy could be useful for other PPI drug discovery attempts.

General Charge Transfer Dipole Model for AMOEBA-Like Force Fields.

The development of highly accurate force fields is always an importance aspect in molecular modeling. In this work, we introduce a general damping-based charge transfer dipole (D-CTD) model to describe the charge transfer energy and the corresponding charge flow for H, C, N, O, P, S, F, Cl, and Br elements in common bio-organic systems. Then, two effective schemes to evaluate the charge flow from the corresponding induced dipole moment between the interacting molecules were also proposed and discussed. The potential applicability of the D-CTD model in ion-containing systems was also demonstrated in a series of ion-water complexes including Li+, Na+, K+, Mg2+, Ca2+, Fe2+, Zn2+, Pt2+, F-, Cl-, Br-, and I- ions. In general, the D-CTD model demonstrated good accuracy and good transferability in both charge transfer energy and the corresponding charge flow for a wide range of model systems. By distinguishing the intermolecular charge redistribution (charge transfer) under the influence of an external electric field from the accompanying intramolecular charge redistribution (polarization), the D-CTD model is theoretically consistent with current induced dipole-based polarizable dipole models and hence can be easily implemented and parameterized. Along with our previous work in charge penetration-corrected electrostatics, a bottom-up approach constructed water model was also proposed and demonstrated. The structure-maker and structure-breaker roles of cations and anions were also correctly reproduced using Na+, K+, Cl-, and I- ions in the new water model, respectively. This work demonstrates a cost-effective approach to describe the charge transfer phenomena. The water and ion models also show the feasibility of a modulated development approach for future force fields.

Reliable Prediction of the Protein-Ligand Binding Affinity Using a Charge Penetration Corrected AMOEBA Force Field: A Case Study of Drug Resistance Mutations in Abl Kinase.

Protein mutations that directly impair drug binding are related to therapeutic resistance, and accurate prediction of their impact on drug binding would benefit drug design and clinical practice. Here, we have developed a scoring strategy that predicts the effect of the mutations on the protein-ligand binding affinity. In view of the critical importance of electrostatics in protein-ligand interactions, the charge penetration corrected AMOEBA force field (AMOEBA_CP model) was employed to improve the accuracy of the calculated electrostatic energy. We calculated the electrostatic energy using an energy decomposition analysis scheme based on the generalized Kohn-Sham (GKS-EDA). The AMOEBA_CP model was validated by a protein-fragment-ligand complex data set (Abl236) constructed from the co-crystal structures of the cancer target Abl kinase with six inhibitors. To predict ligand binding affinity changes upon protein mutation of Abl kinase, we used sampling protocol with multistep simulated annealing to search conformations of mutant proteins. The scoring strategy based on AMOEBA_CP model has achieved considerable performance in predicting resistance for 8 kinase inhibitors across 144 clinically identified point mutations. Overall, this study illustrates that the AMOEBA_CP model, which accurately treats electrostatics through penetration correction, enables the accurate prediction of the mutation-induced variation of protein-ligand binding affinity.

Importance of Three-Body Problems and Protein-Protein Interactions in Proteolysis-Targeting Chimera Modeling: Insights from Molecular Dynamics Simulations.

Proteolysis-targeting chimeras (PROTACs) are a class of bifunctional molecules that can induce the ubiquitin degradation of its target protein by hijacking the E3 ligase to form a target protein-PROTAC-E3 ligase ternary complex. Its underlying principle has inspired the development of a wide range of protein degraders that are similar to or beyond PROTACs in recent years. The formation of the ternary complexes is the key to the success of PROTAC-induced protein degradation. Nevertheless, the lack of effective ternary complex modeling techniques has limited the application of computer-aided drug discovery tools to this emerging and fast developing new land in drug industry. Thus, in this study, we explored the application of the more physically sound molecular dynamics simulation and the molecular mechanics combined with the generalized Born and surface area continuum solvation (MM/GBSA) method to solve the underlying three-body problem in PROTAC modeling. We first verified the accuracy of our approach using a series of known Brd4 BD2 degraders. The calculated binding energy showed a good correlation with the experimental Kd values. The modeling of a unique property, namely, the α value, for PROTACs was also first and accurately performed to our best knowledge. The results also demonstrated the importance of PROTAC-induced protein-protein interactions in its modeling, either qualitatively or quantitatively. Finally, by standing on the success of earlier docking-based approaches, our protocol was also applied as a rescoring function in pose prediction. The results showed a notable improvement in reranking the initial poses generated from a modified Rosetta method, which was reportedly one of the best among a handful of PROTAC modeling approaches available in this field. We hope this work could provide a practical protocol and more insights to study the binding and the design of PROTACs and other protein degraders.

3-Silaazetidine: An Unexplored yet Versatile Organosilane Species for Ring Expansion toward Silaazacycles.

Small-ring silacycles are important organosilane species in main-group chemistry and have found numerous applications in organic synthesis. 3-Silaazetidine, a unique small silacycle bearing silicon and nitrogen atoms, has not been adequately explored due to the lack of a general synthetic scheme and its sensitivity to air. Here, we describe that 3-silaazetidine can be easily prepared in situ from diverse air-stable precursors (RSO2NHCH2SiR12CH2Cl). 3-Silaazetidine shows excellent functional group tolerance in a palladium-catalyzed ring expansion reaction with terminal alkynes, giving 3-silatetrahydropyridines and diverse silaazacycle derivatives, which are promising ring frameworks for the discovery of Si-containing functional molecules.

Synthesis, antiepileptic effects, and structure-activity relationships of α-asarone derivatives: In vitro and in vivo neuroprotective effect of selected derivatives.

In the present study, we compared the antiepileptic effects of α-asarone derivatives to explore their structure-activity relationships using the PTZ-induced seizure model. Our research revealed that electron-donating methoxy groups in the 3,4,5-position on phenyl ring increased antiepileptic potency but the placement of other groups at different positions decreased activity. Besides, in allyl moiety, the optimal activity was reached with either an allyl or a 1-butenyl group in conjugation with the benzene ring. The compounds 5 and 19 exerted better neuroprotective effects against epilepsy in vitro (cell) and in vivo (mouse) models. This study provides valuable data for further exploration and application of these compounds as potential anti-seizure medicines.

Calculation of Transport Parameters Using ab initio and AMOEBA Polarizable Force Field Methods.

The transport properties of chemical species such as coefficients of diffusion, thermal conductivity, and viscosity have been widely used in combustion modeling. Lennard-Jones parameters fitted from the accurate intermolecular potential energy surfaces are crucial to obtain such information. Hence, a fast and accurate energy function is always desired for this purpose. In this study, the quality of a widely used polarizable force field AMOEBA was examined for the interaction between noble gases and n-alkanes. First, the intermolecular energy was compared between AMOEBA, MP2/CBS, MP2/aug'-cc-pVDZ, and QCISD(T)/CBS. The root mean squared error of the original AMOEBA was 10.31 cm-1 against QCISD(T)/CBS for all conformations. This was comparable with the errors of 10.84 and 7.75 cm-1 for MP2/aug'-cc-pVDZ and MP2/CBS, respectively. Further optimizing the van der Waals parameters of noble gases, the error of the force field against QCISD(T)/CBS was reduced to 6.24 cm-1, even better than the MP2/CBS results. Based on the optimized force field parameters, the intermolecular Lennard-Jones parameters were derived using the spherically averaged method and one-dimensional minimization method for a set of (n-alkanes, noble gases) pairs. The discrepancy of the one-dimensional minimization predicted Lennard-Jones collision rates from the tabulated values was typically within 10%, while it could be as large as 20-30% for the spherically averaged method. Additionally, the binary diffusion coefficients were calculated using the present Lennard-Jones parameters. In this case, the parameters derived from the spherically averaged method perform better. The mean unsigned error of the diffusion coefficients is usually within 5%, which is in good agreement with the experimental results. The results demonstrate that the AMOEBA force field can be used to generate the transport parameters systematically.

The development of an Amber-compatible organosilane force field for drug-like small molecules.

As members of the group IVA elements, silicon and carbon have long been thought of as isosteres of each other in drug design. However, the lack of silicon parameters in current main stream force fields hinders the computational study of this important element in drug discovery. Thus, in this study, we attempted to supplement the parameters of organosilanes in the General Amber Force Field (GAFF2). The parameters have been designed following the principles of GAFF2 to make it compatible with the Amber force field family. The accuracy of the parameters was discussed by comparing the pair interaction energy, the liquid properties, and the structures and alchemical binding free energy differences for a set of protein-ligand complexes.

Filling the Gap in Understanding the Mechanism of GABAAR and Propofol Using Computational Approaches.

γ-Aminobutyric acid type-A receptors (GABAARs) play a critical role in neural transmission by mediating the inhibitory neural firing and are the target of many psychiatric drugs. Among them, propofol is one of the most widely used and important general anesthetics in clinics. Recent advances in structural biology revealed the structure of a human GABAAR in both open and closed states. Yet, the detailed mechanism of the receptor and propofol remains to be fully understood. Therefore, in this study, based on the previous successes in structural biology, a variety of computational techniques were applied to fill the gap between previous experimental studies. This study investigated the ion-conducting mechanism of GABAAR, predicted the possible binding mechanism of propofol, and revealed a new motion mechanism of transmembrane domain (TMD) helices. We hope that this study may contribute to future studies on ion-channel receptors, general anesthetics, and drug development.

Alpha-asarone Improves Cognitive Function of APP/PS1 Mice and Reducing Aß42, P-tau and Neuroinflammation, and Promoting Neuron Survival in the Hippocampus.

Alzheimer's disease (AD) is a progressive neurodegenerative disease most often characterized by memory impairment and cognitive decline. Alpha-asarone has been reported to have the potential to treat AD. Our previous studies have found that alpha-asarone improves aged rats' cognitive function by alleviating neuronal excitotoxicity via type A gamma-aminobutyric acid (GABA) receptors. GABA level's change, neuroinflammation, and dysfunctional autophagy are found to be associated with AD. However, the effect of alpha-asarone on cognitive function of APP/PS1 transgenic mice and its underlying mechanism in terms of aggregation of amyloid-ß42 (Aß42) and phosphorylated tau (p-tau), glutamic acid decarboxylase (GAD) level, neuroinflammation, and autophagy are unclear. Accordingly, we attempted to explore whether alpha-asarone improves AD mice's cognitive function and alleviates pathological symptoms by regulating GAD level, inhibiting neuroinflammation, or restore autophagy. We found that alpha-asarone enhanced spatial learning memory and decreased Aß42 and p-tau levels without influencing the GAD level in APP/PS1 transgenic mice. Also, it decreased the GFAP expression and reduced pro-inflammatory cytokines levels, thus alleviating neuroinflammation. Furthermore, alpha-asarone decreased the excess number of autophagosomes and promoted hippocampal neurons' survival. In conclusion, the results confirmed the therapeutic effect of alpha-asarone on AD-related astrogliosis, dysfunctional autophagy, and neuronal damage, which indicates its great potential to treat AD.

The application of the MM/GBSA method in the binding pose prediction of FGFR inhibitors.

The success of a structure-based drug is highly dependent on a known binding pose of the protein-ligand system. However, this is not always available. In this study, we set out to explore the applicability of the popular and easy-to-use MD-based MM/GBSA method to determine the binding poses of known FGFR inhibitors. It was found that MM/GBSA combined with 100 ns of MD simulation significantly improved the success rate of docking methods from 30-40% to 70%. This work demonstrates a way that the MM/GBSA method can be more accurate than it is in ligand ranking, filling a gap in structure-based drug discovery when the binding pose is unknown.

Alpha-asarone improves cognitive function of aged rats by alleviating neuronal excitotoxicity via GABAA receptors.

Alzheimer's disease (AD), the most common form of dementia, still lacks effective treatment at present. Alpha-asarone (ASA) is the major compound isolated from the Chinese medicinal herb Acorus gramineus. It has been reported to enhance cognitive function in rodent models, yet its mechanism was not fully understood. In this work, we demonstrated that ASA improved the spatial memory, reduced the neuronal injury, and decreased the level of Aß1-42 in the hippocampus of aged rats. The results also showed that ASA had the neuroprotective effects against glutamate toxicity and decreased cytoplasmic calcium level in primary hippocampal neurons. By comparing the multiple properties of ASA and propofol (PPF) via computer modelling, we speculated that ASA may bind to the PPF binding site of type A gamma (γ)-aminobutyric acid receptors (GABAARs). This was further supported by the whole-cell patch-clamp recording. Our results suggested that ASA, as a GABAAR positive allosteric modulator (PAM), can improve cognitive function of aged rats by alleviating the neuronal overexcitation. Furthermore, the binding mode of ASA on GABAAR may lay a foundation for structure-based drug design in AD therapy.

Practical Synthesis of Benzimidazo[1,2-a]quinolines via Rh(III)-Catalyzed C-H Activation Cascade Reaction from Imidamides and Anthranils.

We report a novel and practical one-pot Rh(III)-catalyzed strategy to construct benzimidazo[1,2-a]quinolines from readily available imidamides and anthranils. The cascade reaction proceeds via a C-H amination-cyclization-cyclization process in ionic liquid without any additives and possesses simple operation, moderate-to-high yield, and broad substrate scope features, which will provide the reference for the construction of biologically active fused benzimidazoles.

Iridium-Catalyzed B-H Bond Insertion Reactions Using Sulfoxonium Ylides as Carbene Precursors toward α-Boryl Carbonyls.

An iridium-catalyzed B-H bond insertion reaction between borane adducts and sulfoxonium ylides to afford α-boryl carbonyls has been developed. The starting materials are safe and readily available. In addition, analogues of sulfoxonium ylides, such as sulfonium salts and sulfonium ylides could also be amenable to the reaction.

Water-mediated C-H activation of arenes with secure carbene precursors: the reaction and its application.

A water-mediated C-H activation using sulfoxonium ylides is reported, providing a general, green and step-economic approach to construct a C-C bond and varieties of useful N-heterocycle scaffolds. Notably, the "water-mediated" activation, in contrast to that in organic solvents, shows great potential in pharmaceutical, biochemistry and chemical industries.