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.

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.

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.

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.

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.

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.

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.

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.

Estimating and modeling charge transfer from the SAPT induction energy.

Recent studies using quantum mechanics energy decomposition methods, for example, SAPT and ALMO, have revealed that the charge transfer energy may play an important role in short ranged inter-molecular interactions, and have a different distance dependence comparing with the polarization energy. However, the charge transfer energy component has been ignored in most current polarizable or non-polarizable force fields. In this work, first, we proposed an empirical decomposition of SAPT induction energy into charge transfer and polarization energy that mimics the regularized SAPT method (ED-SAPT). This empirical decomposition is free of the divergence issue, hence providing a good reference for force field development. Then, we further extended this concept in the context of AMOEBA polarizable force field, proposed a consistent approach to treat the charge transfer phenomenon. Current results show a promising application of this charge transfer model in future force field development. © 2017 Wiley Periodicals, Inc.

Discovery of a potent inhibitor of MELK that inhibits expression of the anti-apoptotic protein Mcl-1 and TNBC cell growth.

Despite recent advances in molecularly directed therapy, triple negative breast cancer (TNBC) remains one of the most aggressive forms of breast cancer, still without a suitable target for specific inhibitors. Maternal embryonic leucine zipper kinase (MELK) is highly expressed in TNBC, where level of overexpression correlates with poor prognosis and an aggressive disease course. Herein, we describe the discovery through targeted kinase inhibitor library screening, and structure-guided design of a series of ATP-competitive indolinone derivatives with subnanomolar inhibition constants towards MELK. The most potent compound, 17, inhibits the expression of the anti-apoptotic protein Mcl-1 and proliferation of TNBC cells exhibiting selectivity for cells expressing high levels of MELK. These studies suggest that further elaboration of 17 will furnish MELK-selective inhibitors with potential for development in preclinical models of TNBC and other cancers.

Scalable improvement of SPME multipolar electrostatics in anisotropic polarizable molecular mechanics using a general short-range penetration correction up to quadrupoles.

We propose a general coupling of the Smooth Particle Mesh Ewald SPME approach for distributed multipoles to a short-range charge penetration correction modifying the charge-charge, charge-dipole and charge-quadrupole energies. Such an approach significantly improves electrostatics when compared to ab initio values and has been calibrated on Symmetry-Adapted Perturbation Theory reference data. Various neutral molecular dimers have been tested and results on the complexes of mono- and divalent cations with a water ligand are also provided. Transferability of the correction is adressed in the context of the implementation of the AMOEBA and SIBFA polarizable force fields in the TINKER-HP software. As the choices of the multipolar distribution are discussed, conclusions are drawn for the future penetration-corrected polarizable force fields highlighting the mandatory need of non-spurious procedures for the obtention of well balanced and physically meaningful distributed moments. Finally, scalability and parallelism of the short-range corrected SPME approach are addressed, demonstrating that the damping function is computationally affordable and accurate for molecular dynamics simulations of complex bio- or bioinorganic systems in periodic boundary conditions.

General van der Waals potential for common organic molecules.

This work presents a systematic development of a new van der Waals potential (vdW2016) for common organic molecules based on symmetry-adapted perturbation theory (SAPT) energy decomposition. The Buf-14-7 function, as well as Cubic-mean and Waldman-Hagler mixing rules were chosen given their best performance among other popular potentials. A database containing 39 organic molecules and 108 dimers was utilized to derive a general set of vdW parameters, which were further validated on nucleobase stacking systems and testing organic dimers. The vdW2016 potential is anticipated to significantly improve the accuracy and transferability of new generations of force fields for organic molecules.

An optimized charge penetration model for use with the AMOEBA force field.

The principal challenge of using classical physics to model biomolecular interactions is capturing the nature of short-range interactions that drive biological processes from nucleic acid base stacking to protein-ligand binding. In particular most classical force fields suffer from an error in their electrostatic models that arises from an ability to account for the overlap between charge distributions occurring when molecules get close to each other, known as charge penetration. In this work we present a simple, physically motivated model for including charge penetration in the AMOEBA (Atomic Multipole Optimized Energetics for Biomolecular Applications) force field. With a function derived from the charge distribution of a hydrogen-like atom and a limited number of parameters, our charge penetration model dramatically improves the description of electrostatics at short range. On a database of 101 biomolecular dimers, the charge penetration model brings the error in the electrostatic interaction energy relative to the ab initio SAPT electrostatic interaction energy from 13.4 kcal mol-1 to 1.3 kcal mol-1. The model is shown not only to be robust and transferable for the AMOEBA model, but also physically meaningful as it universally improves the description of the electrostatic potential around a given molecule.

The molecular mechanism of eukaryotic elongation factor 2 kinase activation.

Calmodulin (CaM)-dependent eukaryotic elongation factor 2 kinase (eEF-2K) impedes protein synthesis through phosphorylation of eukaryotic elongation factor 2 (eEF-2). It is subject to complex regulation by multiple upstream signaling pathways, through poorly described mechanisms. Precise integration of these signals is critical for eEF-2K to appropriately regulate protein translation rates. Here, an allosteric mechanism comprising two sequential conformations is described for eEF-2K activation. First, Ca(2+)/CaM binds eEF-2K with high affinity (Kd(CaM)(app) = 24 ± 5 nm) to enhance its ability to autophosphorylate Thr-348 in the regulatory loop (R-loop) by > 10(4)-fold (k(auto) = 2.6 ± 0.3 s(-1)). Subsequent binding of phospho-Thr-348 to a conserved basic pocket in the kinase domain potentially drives a conformational transition of the R-loop, which is essential for efficient substrate phosphorylation. Ca(2+)/CaM binding activates autophosphorylated eEF-2K by allosterically enhancing k(cat)(app) for peptide substrate phosphorylation by 10(3)-fold. Thr-348 autophosphorylation results in a 25-fold increase in the specificity constant (k(cat)(app)/K(m)(Pep-S) (app)), with equal contributions from k(cat)(app) and K(m)(Pep-S)(app), suggesting that peptide substrate binding is partly impeded in the unphosphorylated enzyme. In cells, Thr-348 autophosphorylation appears to control the catalytic output of active eEF-2K, contributing more than 5-fold to its ability to promote eEF-2 phosphorylation. Fundamentally, eEF-2K activation appears to be analogous to an amplifier, where output volume may be controlled by either toggling the power switch (switching on the kinase) or altering the volume control (modulating stability of the active R-loop conformation). Because upstream signaling events have the potential to modulate either allosteric step, this mechanism allows for exquisite control of eEF-2K output.

United polarizable multipole water model for molecular mechanics simulation.

We report the development of a united AMOEBA (uAMOEBA) polarizable water model, which is computationally 3-5 times more efficient than the three-site AMOEBA03 model in molecular dynamics simulations while providing comparable accuracy for gas-phase and liquid properties. In this coarse-grained polarizable water model, both electrostatic (permanent and induced) and van der Waals representations have been reduced to a single site located at the oxygen atom. The permanent charge distribution is described via the molecular dipole and quadrupole moments and the many-body polarization via an isotropic molecular polarizability, all located at the oxygen center. Similarly, a single van der Waals interaction site is used for each water molecule. Hydrogen atoms are retained only for the purpose of defining local frames for the molecular multipole moments and intramolecular vibrational modes. The parameters have been derived based on a combination of ab initio quantum mechanical and experimental data set containing gas-phase cluster structures and energies, and liquid thermodynamic properties. For validation, additional properties including dimer interaction energy, liquid structures, self-diffusion coefficient, and shear viscosity have been evaluated. The results demonstrate good transferability from the gas to the liquid phase over a wide range of temperatures, and from nonpolar to polar environments, due to the presence of molecular polarizability. The water coordination, hydrogen-bonding structure, and dynamic properties given by uAMOEBA are similar to those derived from the all-atom AMOEBA03 model and experiments. Thus, the current model is an accurate and efficient alternative for modeling water.

Reversible covalent inhibition of eEF-2K by carbonitriles.

eEF-2K is a potential target for treating cancer. However, potent specific inhibitors for this enzyme are lacking. Previously, we identified 2,6-diamino-4-(2-fluorophenyl)-4H-thiopyran-3,5-dicarbonitrile (DFTD) as an inhibitor of eEF-2K. Here we describe its mechanism of action against eEF-2K, on the basis of kinetic, mutational, and docking studies, and use chemoinformatic approaches to identify a similar class of carbonitrile-containing compounds that exhibit the same mechanism of action. We show that DFTD behaves as a reversible covalent inhibitor of eEF-2K with a two-step mechanism of inhibition: a fast initial binding step, followed by a slower reversible inactivation step. Molecular docking suggests that a nitrile group of DFTD binds within 4.5 Å of the active site Cys146 to form a reversible thioimidate adduct. Because Cys146 is not conserved amongst other related kinases, targeting this residue holds promise for the development of selective covalent inhibitors of eEF-2K.

Identification and validation of novel PERK inhibitors.

PERK, as one of the principle unfolded protein response signal transducers, is believed to be associated with many human diseases, such as cancer and type-II diabetes. There has been increasing effort to discover potent PERK inhibitors due to its potential therapeutic interest. In this study, a computer-based virtual screening approach is employed to discover novel PERK inhibitors, followed by experimental validation. Using a focused library, we show that a consensus approach, combining pharmacophore modeling and docking, can be more cost-effective than using either approach alone. It is also demonstrated that the conformational flexibility near the active site is an important consideration in structure-based docking and can be addressed by using molecular dynamics. The consensus approach has further been applied to screen the ZINC lead-like database, resulting in the identification of 10 active compounds, two of which show IC50 values that are less than 10 µM in a dose-response assay.

Synthesis and biological evaluation of pyrido[2,3-d]pyrimidine-2,4-dione derivatives as eEF-2K inhibitors.

A small molecule library of pyrido[2,3-d]pyrimidine-2,4-dione derivatives 6-16 was synthesized from 6-amino-1,3-disubstituted uracils 18, characterized, and screened for inhibitory activity against eukaryotic elongation factor-2 kinase (eEF-2K). To understand the binding pocket of eEF-2K, structural modifications of the pyrido[2,3-d]pyrimidine were made at three regions (R(1), R(2), and R(3)). A homology model of eEF-2K was created, and compound 6 (A-484954, Abbott laboratories) was docked in the catalytic domain of eEF-2K. Compounds 6 (IC50=420nM) and 9 (IC50=930nM) are found to be better molecules in this preliminary series of pyrido[2,3-d]pyrimidine analogs. eEF-2K activity in MDA-MB-231 breast cancer cells is significantly reduced by compound 6, to a lesser extent by compound 9, and is unaffected by compound 12. Similar inhibitory results are observed when eEF-2K activity is stimulated by 2-deoxy-d-glucose (2-DOG) treatment, suggesting that compounds 6 and 9 are able to inhibit AMPK-mediated activation of eEF-2K to a notable extent. The results of this work will shed light on the further design and optimization of novel pyrido[2,3-d]pyrimidine analogs as eEF-2K inhibitors.

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.