Inhibitor Affinity Research Articles

MM/GBSA prediction of relative binding affinities of carbonic anhydrase inhibitors: effect of atomic charges and comparison with Autodock4Zn

Carbonic anhydrase is an attractive drug target for the treatment of many diseases. This paper examines the ability of end-state MM/GBSA methods to rank inhibitors of carbonic anhydrase in terms of their binding affinities. The MM/GBSA binding energies were evaluated using different atomic charge schemes (Mulliken, ESP and NPA) at different levels of theories, including Hartree–Fock, B3LYP-D3(BJ), and M06-2X with the 6–31G(d,p) basis set. For a large test set of 32 diverse inhibitors, the use of B3LYP-D3(BJ) ESP atomic charges yielded the strongest correlation with experiment (R2 = 0.77). The use of the recently enhanced Autodock Vina and zinc optimised AD4Zn force field also predicted ligand binding affinities with moderately strong correlation (R2 = 0.64) at significantly lower computational cost. However, the docked poses deviate significantly from crystal structures. Overall, this study demonstrates the applicability of docking to estimate ligand binding affinities for a diverse range of CA inhibitors, and indicates that more theoretically robust MM/GBSA simulations show promise for improving the accuracy of predicted binding affinities, as long as a validated set of parameters is used.Graphical abstract

Discovery of novel HPPD inhibitors: Virtual screening, molecular design, structure modification and biological evaluation

4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD, a Fe(II)/α-ketoglutarate dependent oxygenases), is a popular herbicide target. In this work, two pharmacophore models based on common molecular characteristics (HipHop) and receptor-ligand complex (CBP) were generated for virtual screening for HPPD inhibitors. About 1,000,000 molecules containing diketone structure from PubChem were filtered by Lipinski's rules to build a 3D database. Then the database was screened through combining HipHop model, CBP model, ADMET (absorption, distribution, metabolism, excretion and toxicity) prediction and molecular docking. Subsequently, based on the specific binding mode and affinity of HPPD inhibitors, 4 molecules with high -CDOCKER energy, good aqueous solubility and human safety predicative properties values were screened. From the screening results and combined with previous work, three novel HPPD inhibitors were designed and synthesized through fragment splicing and bioisosterism strategies. Compound IV-a exhibited similar inhibition of Arabidopsis thaliana HPPD (AtHPPD) and herbicidal activity as mesotrione. Crop selectivity showed that compound IV-a had better crop safety than mesotrione. Comparing the molecular properties, ADMET and molecular docking studies indicated that compounds IV-a exhibited better properties than mesotrione, which could be further modified as novel HPPD inhibitor herbicides.

Characterization of substrates and inhibitors of the human heterodimeric transporter 4F2hc-LAT1 using purified protein and the scintillation proximity radioligand binding assay.

Amino acids have diverse and essential roles in many cellular functions such as in protein synthesis, metabolism and as precursors of different hormones. Translocation of amino acids and derivatives thereof across biological membranes is mediated by amino acid transporters. 4F2hc-LAT1 is a heterodimeric amino acid transporter that is composed of two subunits belonging to the SLC3 (4F2hc) and SLC7 (LAT1) solute carrier families. The ancillary protein 4F2hc is responsible for the correct trafficking and regulation of the transporter LAT1. Preclinical studies have identified 4F2hc-LAT1 as a valid anticancer target due to its importance in tumor progression. The scintillation proximity assay (SPA) is a valuable radioligand binding assay that allows the identification and characterization of ligands of membrane proteins. Here, we present a SPA ligand binding study using purified recombinant human 4F2hc-LAT1 protein and the radioligand [3H]L-leucine as tracer. Binding affinities of different 4F2hc-LAT1 substrates and inhibitors determined by SPA are comparable with previously reported K m and IC 50 values from 4F2hc-LAT1 cell-based uptake assays. In summary, the SPA is a valuable method for the identification and characterization of ligands of membrane transporters including inhibitors. In contrast to cell-based assays, where the potential interference with other proteins such as endogenous transporters persists, the SPA uses purified protein making target engagement and characterization of ligands highly reliable.

Molecular Insight into Drug Resistance Mechanism Conferred by Aberrant PIK3CD Splice Variant in African American Prostate Cancer.

Targeting PI3Kδ has emerged as a promising therapy for hematologic and non-hematologic malignancies. Previously, we identified an oncogenic splice variant, PIK3CD-S, conferring Idelalisib resistance in African American (AA) prostate cancer (PCa). In the current study, we employed a comprehensive analysis combining molecular biology, biochemistry, histology, in silico simulation, and in vitro functional assays to investigate the PIK3CD-S expression profiles in PCa samples and to elucidate the drug resistance mechanism mediated by PI3Kδ-S (encoded by PIK3CD-S). The immunohistochemistry, RT-PCR, and Western blot assays first confirmed that PI3Kδ-S is highly expressed in AA PCa. Compared with PCa expressing the full-length PI3Kδ-L, PCa expressing PI3Kδ-S exhibits enhanced drug resistance properties, including a higher cell viability, more antiapoptotic and invasive capacities, and constitutively activated PI3K/AKT signaling, in the presence of PI3Kδ/PI3K inhibitors (Idelalisib, Seletalisib, Wortmannin, and Dactolisib). Molecular docking, ATP-competitive assays, and PI3 kinase assays have further indicated a drastically reduced affinity of PI3Kδ inhibitors with PI3Kδ-S vs. PI3Kδ-L, attributed to the lack of core binding residues in the PI3Kδ-S catalytic domain. Additionally, SRSF2 has been identified as a critical splicing factor mediating exon 20 skipping in PIK3CD pre-mRNA. The inhibition of the SRSF2 activity by SRPIN340 successfully sensitizes AA PCa cells to PI3Kδ inhibitors, suggesting a novel therapeutic option for Idelalisib-resistant tumors.

Structure of pyridoxal 5'-phosphate-bound D-threonine aldolase from Chlamydomonas reinhardtii.

D-Threonine aldolase (DTA) is a pyridoxal-5'-phosphate-dependent enzyme which catalyzes the reversible aldol reaction of glycine with a corresponding aldehyde to yield the D-form β-hydroxy-α-amino acid. This study produced and investigated the crystal structure of DTA from Chlamydomonas reinhardtii (CrDTA) at 1.85 Å resolution. To our knowledge, this is the first report on the crystal structure of eukaryotic DTA. Compared with the structure of bacterial DTA, CrDTA has a similar arrangement of active-site residues. On the other hand, we speculated that some non-conserved residues alter the affinity for substrates and inhibitors. The structure of CrDTA could provide insights into the structural framework for structure-guided protein engineering studies to modify reaction selectivity.

Potential Health Benefits of Banana Phenolic Content during Ripening by Implementing Analytical and In Silico Techniques.

Banana ranks as the fifth most cultivated agricultural crop globally, highlighting its crucial socio-economic role. The banana's health-promoting benefits are correlated with its composition in bioactive compounds, such as phenolic compounds. Thus, the present study attempts to evaluate the potential health benefits of banana phenolic content by combing analytical and in silico techniques. Particularly, the total phenolic content and antioxidant/antiradical activity of banana samples during ripening were determined spectrophotometrically. In parallel, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was implemented to unravel the variations in the phenolic profile of banana samples during ripening. Chlorogenic acid emerged as a ripening marker of banana, while apigenin and naringenin were abundant in the unripe fruit. In a further step, the binding potential of the elucidated phytochemicals was examined by utilizing molecular target prediction tools. Human carbonic anhydrase II (hCA-II) and XII (hCA-XII) enzymes were identified as the most promising targets and the inhibitory affinity of phenolic compounds was predicted through molecular docking studies. This class of enzymes is linked to a variety of pathological conditions, such as edema, obesity, hypertension, cancer, etc. The results assessment indicated that all assigned phenolic compounds constitute great candidates with potential inhibitory activity against CA enzymes.

Approaching the Dimerization Mechanism of Small Molecule Inhibitors Targeting PD-L1 with Molecular Simulation.

Inhibitors blocking the PD-1/PD-L1 immune checkpoint demonstrate impressive anti-tumor immunity, and small molecule inhibitors disclosed by the Bristol-Myers Squibb (BMS) company have become a hot topic. In this work, by modifying the carbonyl group of BMS-202 into a hydroxyl group to achieve two enantiomers (MS and MR) with a chiral center, we found that this is an effective way to regulate its hydrophobicity and thus to reduce the negative effect of polar solvation free energy, which enhances the stability of PD-L1 dimer/inhibitor complexes. Moreover, we studied the binding modes of BMS-200 and BMS-202-related small molecule inhibitors by molecular dynamics simulation to explore their inhibitory mechanism targeting PD-L1 dimerization. The results showed that the size exclusion effect of the inhibitors triggered the rearrangement of the residue ATyr56, leading to the formation of an axisymmetric tunnel-shaped pocket, which is an important structural basis for improving the binding affinity of symmetric inhibitors with PD-L1. Furthermore, after inhibitor dissociation, the conformation of ATyr123 and BMet115 rearranged, which blocked the entrance of the binding pocket, while the reverse rearrangements of the same residues occurred when the PD-L1 monomer was complexed with the inhibitors, preparing PD-L1 for dimerization. Overall, this study casts a new light on the inhibitory mechanism of BMS inhibitors targeting PD-L1 dimerization and provides an idea for designing novel small molecule inhibitors for future cancer immunotherapy.

Synthesis of amantadine clubbed N-aryl amino thiazoles as potent urease, α-amylase & α-glucosidase inhibitors, kinetic and molecular docking studies.

A series of ten novel compounds were synthesized by incorporating a 1,3 thiazole core into amantadine and their structures were validated using different analytical and spectral methods such as FTIR, EI-MS, 1H NMR, and 13C NMR. The antibacterial and enzyme inhibitory properties of these newly synthesized compounds were evaluated. Remarkably, the compounds exhibited significant antibacterial activity against Escherichia coli and Bacillus subtilis. Additionally, the in vitro inhibitory activities of the synthesized compounds, against α-amylase, α-glucosidase, and urease were investigated. Among the tested compounds, compound 6d demonstrated potent and selective inhibition of α-amylase IC50 = 97.37 ± 1.52 μM, while acarbose was used as positive control and exhibited IC50 = 5.17 ± 0.25 μM. Compound 6d and 6e exhibited prominent inhibition against α-glucosidase IC50 = 38.73 ± 0.80 μM and 41.63 ± 0.26 μM respectively. Furthermore, compound 6d inhibited urease with exceptional efficacy IC50 = 32.76 μM, while positive control thiourea showed more prominent activity having IC50 = 1.334 μM. Molecular docking studies disclosed the binding mechanism and affinity of these new inhibitors within the binding sites of various amino acids. To investigate the association between molecular structural characteristics and inhibitory actions of synthesized derivatives, preliminary structure-activity relationship (SAR) studies were performed. These findings indicated that compounds 6a, 6c, 6d and 6e are potential candidates for hit-to-lead follow-up in the drug-discovery process for treating diabetes and hyperglycemia.

Dynamics and binding affinity of nucleoside and non-nucleoside inhibitors with RdRp of SARS-CoV-2: a molecular screening, docking, and molecular dynamics simulation study

In this COVID-19 pandemic situation, an appropriate drug is urgent to fight against this infectious disease to save lives and prevent mortality. Repurposed drugs and vaccines are the immediate solutions for this medical emergency until discover a new drug to treat this disease. As of now, no specific drug is available to cure this disease completely. Several drug targets were identified in SARS-CoV-2, in which RdRp protein is one of the potential targets to inhibit this virus infection. In-Silico studies plays a vital role to understand the binding nature of the drugs at the atomic level against the disease targets. The present study explores the binding mechanism of reported 53 nucleoside and non-nucleoside RdRp inhibitors and Ivermectin which are in clinical trials. These molecules were screened by molecular docking simulation; in which, the molecules are showing high binding affinity and forming interactions with the key amino acids of active site of RdRp protein are chosen for molecular dynamics simulation (MD) and binding free energy analysis. The results of molecular docking and MD simulation studies reveal that IDX184 is a stable molecule and forms strong interactions with the key amino acids and shows high binding affinity towards RdRp. Hence, IDX184 may also be considered as a potential inhibitor of RdRp after clinical study. Communicated by Ramaswamy H. Sarma

Analysis of Molecular Docking Chemical Content of Lime (Citrus aurantiifolia (Christm.) Swingle) Against Diabetes Mellitus Therapy Targets and Prediction of Pharmacokinetic Profiles and Toxicity

Diabetes mellitus drugs currently available are sulfonylureas, biguanides, thiazolidines, and alpha glucoside inhibitors which are widely used to control hyperglycemia. These drugs cannot prevent complications of diabetes, and these drugs should not be used continuously because it causes undesirable pathological conditions. The essential oil in lime peel is rich in phenolics, especially flavonoids, which can prevent oxidative stress. This research was carried out computationally to determine the affinity of the compound mechanism in lime, pharmacokinetic profile, and toxicity of the chemical content of lime which is thought to have antihyperglycemic activity using chemoinformatics studies. The hardware used is an Asus laptop X441UB-GA502T Intel Core I5 – 8250 DDR 4 4GB HD 1TB VGA mix 110 2GB screen 14 "DVD-RW WIN 10 ORI. The software used is PLANTS (PROTEIN-LIGAND ANT SYSTEM), YASARA, Marvin sketch, Swisstargetprediction, SwissADME, and Toxtree. All compounds in lime were most active against the target protein PPARγ with an average value of -78.0092, while the positive control value of thiazolidinediones was -90.3393. The highest inhibitory affinity of the compound contained in lime was hesperidin with the target protein DPP-4 of -113.614, higher than the positive control sitagliptin with an inhibitory affinity value of -107.591. Hesperidin absorption in the digestive tract is low, the topology polar surface area (TPSA) value is 234.29Å2, and low polarity and high lipophilicity. There are unexpected heterocyclic compounds, so it becomes a warning against the potential for genotoxic carcinogenicity, namely oxygen element "o".

Pose, duplicate, then elaborate: Steps towards increased affinity for inhibitors targeting the specificity surface of the Pim-1 kinase

In this study, fragment-sized hits binding to Pim-1 kinase with initially modest affinity were further optimized by combining computational, synthetic and crystallographic expertise, eventually resulting in potent ligands with affinities in the nanomolar range that address rarely-targeted regions of Pim-1 kinase. Starting from a set of crystallographically validated, chemically distinct fragments that bind to Pim-1 kinase but lack typical nucleotide mimetic structures, a library of extended fragments was built by exhaustive in silico reactions. After docking, minimization, clustering, visual inspection of the top-ranked compounds, and evaluation of ease of synthetic accessibility, either the original compound or a close derivative was synthesized and tested against Pim-1. For compounds showing the highest degree of Pim-1 inhibition the binding mode was determined crystallographically. Following a structure-guided approach, these were further optimized in a subsequent design cycle improving the compound's initial affinity by several orders of magnitude while synthesizing only a comparatively modest number of derivatives. The combination of computational and experimental approaches resulted in the development of a reasonably potent, novel molecular scaffold for inhibition of Pim-1 that targets specific surface regions, such as the interaction with R122 and P123 of the hinge region, which has been less frequently investigated in similar studies.

Affinity probes based on small-molecule inhibitors for tumor imaging.

Methods for molecular imaging of target areas, including optical imaging, radionuclide imaging, magnetic resonance imaging and other imaging technologies, are helpful for the early diagnosis and precise treatment of cancers. In addition to cancer management, small-molecule inhibitors are also used for developing cancer target probes since they act as the tight-binding ligands of overexpressed proteins in cancer cells. This review aims to summarize the structural designs of affinity probes based on small-molecule inhibitors from the aspects of the inhibitor, linker, dye and radionuclide, and discusses the influence of the modification of these structures on affinity and pharmacokinetics. We also present examples of inhibitor affinity probes in clinical applications, and these summaries will provide insights for future research and clinical translations.

Calculation of binding affinity of JAK1 inhibitors via accurately computational estimation

Janus kinase 1 (JAK1) is a tyrosine kinase that is involved in the initiation of responses to a number of different cytokine receptor families. The JAK1-dependent pathway is a therapeutic target, and several JAK inhibitors have been developed thanks to intensive research. However, since the ATP binding sites of JAK family members are quite alike, JAK1 inhibitors can thus be less selective, resulting in unanticipated adverse effects. Despite this, minor variations in the ATP-binding site have been extensively used to find a variety of small compounds with different inhibitory properties. Stronger binding affinity of JAK1 inhibitors is believed to be able to reduce the negative effects, leading to better treatment results. Therefore, a thorough computational search that can effectively identify ligands with extremely high binding affinity for JAK1 to serve as promising inhibitors is required. Here, a method combining steered-molecular dynamic (SMD) simulations with a modified linear interaction energy (LIE) model has been developed to evaluate the binding affinities of known JAK1 inhibitors. The correlation coefficient between the estimated and experimental values was 0.72 and a root-mean-square error was 0.97 kcal•mol−1, revealing that the SMD/LIE method can precisely and quickly predict the binding free energies of JAK1 inhibitors. Furthermore, three marine fungus-derived compounds, namely hansforesters E, hansforesters G and tetroazolemycins B, were identified to be particularly promising JAK1 inhibitors, accordingly. These findings show that the SMD/LIE method has a lot of promise for in silico screening of possible JAK1 inhibitors from a vast number of compounds that are now accessible. Communicated by Ramaswamy H. Sarma

Computational Design of Miniprotein Inhibitors Targeting SARS-CoV-2 Spike Protein.

The ongoing pandemic of COVID-19 caused by SARS-CoV-2 has become a global health problem. There is an urgent need to develop therapeutic drugs, effective therapies, and vaccines to prevent the spread of the virus. The virus first enters the host cell through the interaction between the receptor binding domain (RBD) of spike protein and the peptidase domain (PD) of the angiotensin-converting enzyme 2 (ACE2). Therefore, blocking the binding of RBD and ACE2 is a promising strategy to inhibit the invasion and infection of the virus in the host cell. In the study, we designed several miniprotein inhibitors against SARS-CoV-2 by single/double/triple-point mutant, based on the initial inhibitor LCB3. Molecular dynamics (MD) simulations and trajectory analysis were performed for an in-depth analysis of the structural stability, essential protein motions, and per-residue energy decomposition involved in the interaction of inhibitors with the RBD. The results showed that the inhibitors have adapted the protein RBD in the binding interface, thereby forming stable complexes. These inhibitors display low binding free energy in the MM/PBSA calculations, substantiating their strong interaction with RBD. Moreover, the binding affinity of the best miniprotein inhibitor, H6Y-M7L-L17F mutant, to RBD was ∼45 980 times (ΔG = RT ln Ki) higher than that of the initial inhibitor LCB3. Following H6Y-M7L-L17F mutant, the inhibitors with strong binding activity are successively H6Y-L17F, L17F, H6Y, and F30Y mutants. Our research proves that the miniprotein inhibitors can maintain their secondary structure and have a highly stable blocking (binding) effect on SARS-CoV-2. This study proposes novel miniprotein mutant inhibitors with enhanced binding to spike protein and provides potential guidance for the rational design of new SARS-CoV-2 spike protein inhibitors.

Abstract 379: Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells

Abstract NanoBRET࣪ target engagement (TE) is the first biophysical technique that broadly enables the quantitative determination of protein inhibitor occupancy in live cells, without disruption of cellular membrane integrity. This quantitative capability is achieved in live cells via BRET with an optimized set of cell-permeable tracers, allow the measurement of compound binding to a selected cellular target protein. RAS is a well-known oncogene that is frequently mutated in most lung, pancreatic and colorectal cancers and is associated with poor disease prognosis. Mutated RAS is locked in the activated GTP bound state and facilitates enhanced RAS signaling in cancer cells. While being a desirable target, the absence of good druggable binding pockets has made modulator compound discovery challenging and unsuccessful. The recent discovery of a unique switch II binding pocket and successful inhibition of the KRAS (G12C) mutant by covalent inhibitors have led to the resurgence of interest in the design of inhibitors targeting RAS directly. Here, we performed NanoBRET࣪ TE cellular assay with RAS inhibitors against transfected RAS and its mutants. Our data demonstrate that NanoBRET࣪ TE cellular assay can measure the apparent affinity of RAS inhibitors by competitive displacement of a NanoBRET࣪ RAS switch I/II pocket tracer, reversibly bound to the LgBiT- and SmBiT- KRAS, KRAS (G12C), KRAS (G12D), KRAS (G12V), KRAS (G13D), KRAS (Q61H), KRAS (Q61L), KRAS (Q61R), or HRAS fusion constructs co-transfected in live HEK293 cells. Z factor analysis indicates the assay is High Throughput Screening (HTS) compatible and reproducible. In addition, we are able to confirm the signaling inhibition of downstream phospho-ERK1/2 by KRAS (G12C)-specific inhibitors AMG 510 (Sotorasib), ARS-1620, MRTX849, and MRTX1257 in MIA PaCa-2 pancreas and SW837 colon cancer cells bearing KRAS (G12C) mutation by Western blot assay. Our results suggest NanoBRET࣪ TE cellular assay can serve as a great tool to facilitate RAS pathway drug discovery against human cancers. Citation Format: Jianghong Wu, Shawn McGinley, Yuan Wang, Peter Gallagher, Haiching Ma. Application of NanoBRET target engagement cellular assay for measurement of inhibitor binding to wild type and mutant RAS in live cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 379.

A computationally affordable approach for accurate prediction of the binding affinity of JAK2 inhibitors.

Janus kinase 2 (JAK2) inhibitors are potential anticancer drugs in the treatment of lymphoma, leukemia, thrombocytosis and particularly myeloproliferative diseases.However, the resemblance among JAK family members has challenged the identification of highly selective inhibitors for JAK2 to reduce undesired side effects. As a result, a robust search for promising JAK2 inhibitors using a computational approach that can effectively nominate new potential candidates to be further analyzed through laborious experimental operations has become necessary. In this study, the binding affinities of JAK2 inhibitors were rapidly and precisely estimated using the fast pulling of ligand (FPL) simulations combined with a modified linear interaction energy (LIE) method. The approach correlates with the experimental binding affinities of JAK2 inhibitors with a correlation coefficient of R = 0.82 and a root-mean-square error of 0.67kcal•mol-1. The data reveal that the FPL/LIE method is highly approximate in anticipating the relative binding free energies of known JAK2 inhibitors with an affordable consumption of computational resources, and thus, it is very promising to be applied in in silico screening for new potential JAK2 inhibitors from a large number of molecules available.

Model-based characterization of permeability damage control through inhibitor injection under parametric uncertainty

Damage in subsurface formations caused by mineral precipitation decreases the porosity and permeability, eventually reducing the production rate of wells in plants producing oil, gas or geothermal fluids. A possible solution to this problem consists in stopping the production followed by the injection of inhibiting species that slow down the precipitation process. In this work we model inhibitor injection and quantify the impact of a set of model parameters on the outputs of the system. The parameters investigated concern three key factors contributing to the success of the treatment: i) the inhibitor affinity, described by an adsorption Langmuir isotherm, ii) the concentration and time related to the injection and iii) the efficiency of the inhibitor in preventing mineral precipitation. Our simulations are set in a stochastic framework where these inputs are characterized in probabilistic terms. Forward simulations rely on a purpose-built code based on finite differences approximation of the reactive transport setup in radial coordinates. We explore the sensitivity diverse outputs, encompassing the well bottom pressure and space-time scales characterizing the transport of the inhibitor. We find that practically relevant output variables, such as inhibitor lifetime and well bottom pressure, display a diverse response to input uncertainties and display poor mutual dependence. Our results quantify the probability of treatment failure for diverse scenarios of inhibitor-rock affinity. We find that treatment optimization based on single outputs may lead to high failure probability when evaluated in a multi-objective framework. For instance, employing an inhibitor displaying an appropriate lifetime may fail in satisfying criteria set in terms of well-bottom pressure history or injected inhibitor mass.

Density functional studies and spectroscopic analysis (FT-IR, FT-Raman, UV–visible, and NMR) with molecular docking approach on an anticancer and antifungal drug 4‑hydroxy-3-methoxybenzaldehyde

4‑hydroxy-3-methoxybenzaldehyde was investigated quantum chemically by DFT approach, surface analysis by Hirshfeld and experimentally by NMR (FT-IR, UV-Visible 1HNMR and 13CNMR). The B3LYP method and the 6–311 ++ G (d, p) basis set were applied to optimize the molecular structure and to calculate the wavenumbers of normal vibrational modes. A detailed description of the intermolecular interactions of the crystal surface was carried out by means of Hirshfeld surface analysis and fingerprint plots found. The calculated bond lengths and angles were compared with the experimental bond lengths and bond angle parameters and found to be in good agreement. VEDA successfully carried out for the distribution of potential energy. 1HNMR and 13CNMR shifts were estimated using the GIAO method and the results compared with experimental spectra. The molecule reactivity region MEP, molecular stability, NBO, and HOMO-LUMO taken into account. Highest occupied and lowest unoccupied energies were computed and found to be -6.413 eV and -1.873 eV, respectively, indicating charge transfer inside the molecule. Chemical descriptors indicate the reactivity of the molecule as a whole, and Fukui function calculations were used to examine the reactive locations of the compound. NBO analysis indicates that the greatest second order perturbation energy E(2) = 31.26 kcal/mol associated with electron delocalization from the donor LP(2) of O2 → π*(C1-C3) acceptor interaction. Many of the proteins for ligand have been used to examine the biological activity, and the results show that the titled compound may have anti-cancer, antioxidant, and antibacterial activities. The drug-likeness was also studied and molecular docking was done using different proteins and with binding energy -5.9. This system has also been subjected to docking and molecular dynamic simulations in order to better visualize binding sites and the effect of ligand on 7POM conformation. The binding free energy of the receptor protein complex was computed to revalidate the inhibitor affinity for the receptor protein complex predicted by docking and molecular dynamic simulation studies. Binding free energy of docked complex estimated by using MM/PBSA approach.

CLAffinity: A Software Tool for Identification of Optimum Ligand Affinity for Competition-Based Primary Screens.

A simplistic assumption in setting up a competition assay is that a low affinity labeled ligand can be more easily displaced from a target protein than a high affinity ligand, which in turn produces a more sensitive assay. An often-cited paper correctly rallies against this assumption and recommends the use of the highest affinity ligand available for experiments aiming to determine competitive inhibitor affinities. However, we have noted this advice being applied incorrectly to competition-based primary screens where the goal is optimum assay sensitivity, enabling a clear yes/no binding determination for even low affinity interactions. The published advice only applies to secondary, confirmatory assays intended for accurate affinity determination of primary screening hits. We demonstrate that using very high affinity ligands in competition-based primary screening can lead to reduced assay sensitivity and, ultimately, the discarding of potentially valuable active compounds. We build on techniques developed in our PyBindingCurve software for a mechanistic understanding of complex biological interaction systems, developing the “CLAffinity tool” for simulating competition experiments using protein, ligand, and inhibitor concentrations common to drug screening campaigns. CLAffinity reveals optimum labeled ligand affinity ranges based on assay parameters, rather than general rules to optimize assay sensitivity. We provide the open source CLAffinity software toolset to carry out assay simulations and a video summarizing key findings to aid in understanding, along with a simple lookup table allowing identification of optimal dynamic ranges for competition-based primary screens. The application of our freely available software and lookup tables will lead to the consistent creation of more performant competition-based primary screens identifying valuable hit compounds, particularly for difficult targets.

Water molecule-mediated selective inhibition of bacterial zinc metalloproteinases by non-hydroxamate compounds: Ab initio molecular simulations

UDP-3-O-acyl-N acetylglucosamine deacetylase (LpxC), Zn metalloenzyme for Gram-negative bacteria is an attractive target for developing novel therapeutic agents. Since LpxC has the similar binding pocket as the human matrix metalloproteinases (MMPs), LpxC inhibitors might also inhibit MMP functions producing side effects in human bodies. Here, we investigated specific interactions between LpxC/MMP and their inhibitors using ab initio molecular simulations to elucidate the reason of selective inhibition for LpxC by non-hydroxamate compounds. The evaluated binding properties between LpxC and the compounds are comparable to the trend of their observed inhibitory affinities. It was also elucidated that compound 22 binds most strongly to LpxC due to its specific interactions with Zn ion and Asp241 side chain of LpxC. In contrast, the interactions between the compounds and MMP are significantly weakened due to the water molecules, which are tightly coordinated with the Zn ion in MMP and interrupt the binding of the compounds to the Zn ion. Accordingly, the present molecular simulations revealed that these water molecules around the Zn ion in MMP are causally related to the selective inhibition of these compounds for LpxC rather than MMP.