Drug Binding Research Articles

Can Benzimidazole Resistance Undermine the Philippines’ Success in Controlling and Eliminating Soil-Transmitted Helminth Infections? A Mini-Review

Abstract Soil-transmitted helminth (STH) infections remain a significant global health burden, affecting over 1.5 billion people worldwide. In the Philippines, STH infections continue to be prevalent, despite ongoing control efforts. The Integrated Helminth Control Program (IHCP), whose cornerstone is mass drug administration (MDA) with benzimidazole drugs, has shown some success in reducing STH prevalence in the country. However, the persistence of infection prevalence being above the national and global targets and the potential for benzimidazole resistance have raised concerns about the long-term sustainability of current control strategies. This review examines the development of benzimidazole resistance in STH, focusing on the role of single nucleotide polymorphisms (SNPs) in the β-tubulin isotype that alter the protein’s amino acid constitution, thereby negatively affecting benzimidazole drug binding efficiency. This review discusses the epidemiology of STH infections in the Philippines, the implementation of IHCP and the potential challenges posed by benzimidazole resistance. This review highlights the need for further research to assess the occurrence of benzimidazole resistance in Philippine STH populations and to explore alternative control strategies. Understanding the mechanisms of benzimidazole resistance and developing effective countermeasures is crucial for achieving sustainable STH control and elimination in the Philippines.

Novel FRET-Based Biosensors for Real-Time Monitoring of Estrogen Receptor Dimerization and Translocation Dynamics in Living Cells.

Estrogen receptors (ERs), comprising ER α and ER β, are crucial for regulating cell growth and differentiation via homo- and hetero-dimer formation. However, accurately detecting ER dimerization with precise spatiotemporal resolution remains a significant challenge. In this study, fluorescence resonance energy transfer-based biosensors to monitor ER dynamics in real-time, are developed and optimized.This approach involves comprehensive structural analysis, linker comparison, and the selection of optimal fluorescent protein pairs, resulting in three distinct biosensors capable of detecting all ER homo- and hetero-dimerizations within the nucleus. These biosensors are utilized to reveal interactions between ER α/β and calmodulin during dimer formation.Furthermore, by leveraging the ligand-binding domain (LBD) of ER β, ER ββ LBD biosensor is designed for real-time analysis of ER ββ homodimerization in the cytoplasm, enhancing the ability to screen ER dimerization-related drugs.Additionally, we developed a novel ER ββ translocation biosensor, which enables real-time observation of ER ββ translocation to the nucleus-a capability previously unavailable, is developed. This spatiotemporal analysis demonstrates the relevance of ER translocation in response to drug binding efficacy and extracellular matrix changes. Our biosensors offertransformative tools for studying ER dynamics, providing valuable insights for drug screening and the investigation of ER-related cellular processes.

Molecular Dynamics Unveils Multiple-Site Binding of Inhibitors with Reduced Activity on the Surface of Dihydrofolate Reductase.

The sensitivity to protein inhibitors is altered by modifications or protein mutations, as represented by drug resistance. The mode of stable drug binding to the protein pocket has been experimentally clarified. However, the nature of the binding of inhibitors with reduced sensitivity remains unclear at the atomic level. In this study, we analyzed the thermodynamics and kinetics of inhibitor binding to the surface of wild-type and mutant dihydrofolate reductase (DHFR) using molecular dynamics simulations combined with Markov state modeling. A strong inhibitor of methotrexate (MTX) showed a preference for the active site of wild-type DHFR with minimal binding to unrelated (secondary) sites. Deletion of a side-chain fragment in MTX largely destabilized the active site-bound state, with clear evidence of binding to secondary sites. Similarly, the F31V mutation in DHFR diminished the specificity of MTX binding to the active site. These results reveal the presence of multiple-bound states whose stabilities are comparable to or higher than those of the unbound state, suggesting that a reduction in the binding affinity for the active site significantly elevates the fractions of these states. This study presents a theoretical model that more accurately interprets the altered drug sensitivity than the traditional two-state model.

Structural basis for antibiotic transport and inhibition in PepT2

The uptake and elimination of beta-lactam antibiotics in the human body are facilitated by the proton-coupled peptide transporters PepT1 (SLC15A1) and PepT2 (SLC15A2). The mechanism by which SLC15 family transporters recognize and discriminate between different drug classes and dietary peptides remains unclear, hampering efforts to improve antibiotic pharmacokinetics through targeted drug design and delivery. Here, we present cryo-EM structures of the proton-coupled peptide transporter, PepT2 from Rattus norvegicus, in complex with the widely used beta-lactam antibiotics cefadroxil, amoxicillin and cloxacillin. Our structures, combined with pharmacophore mapping, molecular dynamics simulations and biochemical assays, establish the mechanism of beta-lactam antibiotic recognition and the important role of protonation in drug binding and transport.

SARS-CoV-2 ORF 3a-mediated currents are inhibited by antiarrhythmic drugs.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to cardiovascular complications, notably cardiac arrhythmias. The open reading frame (ORF) 3a of the coronavirus genome encodes for a transmembrane protein that can function as an ion channel. The aim of this study was to investigate the role of the SARS-CoV-2 ORF 3a protein in COVID-19-associated arrhythmias and its potential as a pharmacological target. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and cultured human fibroblasts were infected with SARS-CoV-2. Subsequent immunoblotting assays revealed the expression of ORF 3a protein in hiPSC-CM but not in fibroblasts. After intracytoplasmic injection of RNA encoding ORF 3a proteins into Xenopus laevis oocytes, macroscopic outward currents could be measured. While class I, II, and IV antiarrhythmic drugs showed minor effects on ORF 3a-mediated currents, a robust inhibition was detected after application of class III antiarrhythmics. The strongest effects were observed with dofetilide and amiodarone. Finally, molecular docking simulations and mutagenesis studies identified key amino acid residues involved in drug binding. Class III antiarrhythmic drugs are potential inhibitors of ORF 3a-mediated currents, offering new options for the treatment of COVID-19-related cardiac complications.

Insights into Antibacterial Design: Computational Modeling of Eugenol Derivatives Targeting DNA Gyrase

The rise of antibiotic resistance underscores the urgent need for novel antibacterial agents. DNA gyrase, an essential enzyme involved in bacterial DNA replication, is a promising target for antibacterial therapy. Computational approaches offer a cost-effective means to design and screen potential inhibitors, such as eugenol derivatives. This study aims to computationally design eugenol derivatives as potential antibacterial agents targeting DNA gyrase, assess their binding affinities, evaluate physicochemical properties, and toxicity, and select lead compounds for further investigation. Molecular docking simulations were conducted to investigate the binding affinities of eugenol derivatives and controls to DNA gyrase. Physicochemical properties and toxicity assessments of eugenol were evaluated. Lead compounds were selected based on drug likeness, toxicity, and binding affinity. Molecular docking studies revealed varying binding affinities of eugenol derivatives to DNA gyrase, with lead compounds exhibiting superior affinity compared to eugenol. Physicochemical properties indicated moderate lipophilicity and low aqueous solubility for eugenol. Toxicity assessment revealed mutagenicity and tumorigenicity. De novo compound synthesis generated 244 novel compounds, with 44 selected based on drug-likeness, toxicity, and binding affinity as lead candidates. These findings provide valuable insights for the development of novel antibacterial agents targeting DNA gyrase, with implications for combating antibiotic resistance.

In silico, In vitro, and In vivo Evaluation of the Anti-alzheimer’s Activity of Berberine

Background: Alzheimer’s disease (AD), a progressive neurodegenerative disease for which there is no effective cure is among the leading causes of death worldwide. Objectives: To investigate the potential anti-AD activity of berberine (BBR). Methods: In silico assessment included molecular docking and ADMET prediction. BBR’s in vitro inhibitory activity of the target selected from docking results was assessed via colorimetric inhibitor screening assay. BBR’s LC50 in adult zebrafish was determined via an Acute Toxicity Study. ZnCl2 concentration for AD induction was determined via toxicity study and T-maze test. Finally, zebrafish were treated with ZnCl2 alone or simultaneously with either BBR or donepezil and assessed via the inhibitory avoidance task, followed by ELISA of AD-related biomarker levels in brain tissue. Results: The in silico assessment showed BBR’s desirable drug properties and binding affinity on selected AD-related targets, which was the greatest docking score with AChE. The in vitro IC50 on AChE was 3.45 μM. The LC50 in adult zebrafish was calculated at 366 ppm. In the T-maze test, ZnCl2 at 2.5 ppm caused the greatest cognitive impairment accompanied by moderate freezing. In the inhibitory avoidance test, fish treated with either 100 ppm BBR or 2.5 ppm donepezil had significantly better performance than ZnCl2-treated fish. ZnCl2-treated zebrafish brain tissue had the highest Aβ levels and AChE activity of all groups, but these were significantly lower in donepeziland BBR-treated fish. ZnCl2- and donepezil-treated fish had similar TNF-α levels, whereas BBR treatment significantly lowered them close to those of untreated fish. Conclusion: BBR showed anti-amyloidogenic, anti-AChE, and anti-inflammatory effects, which support its potential use in AD therapy.

Generation of affinity maps for thiazolidinediones with human serum albumin using affinity microcolumns. II. Effects of advanced glycation end products on multisite drug binding

Multisite protein interactions by the thiazolidinedione-class drugs pioglitazone and rosiglitazone were examined by using high-performance affinity microcolumns that contained normal human serum albumin (HSA) vs HSA that had been modified to form advanced glycation end products by glyoxal (Go) or methylglyoxal (MGo). The results were used to generate an affinity map for these drugs at several key regions on HSA. Strong binding (∼105 M−1) by these drugs was seen at both Sudlow sites I and II. About a 50 % decrease in the affinities at Sudlow site II was observed for pioglitazone for Go-modified HSA, while either a 47 % decrease or 1.6-fold increase in affinity was seen for MGo-modified HSA, depending on the extent of modification. The binding affinity for rosiglitazone at Sudlow site II had a 40–83 % decrease for Go-modified HSA and either a non-significant change or 1.4-fold increase for MGo-modified HSA. At Sudlow site I, pioglitazone gave a 41 % decrease in affinity for either Go or MGo-modified HSA, and for rosiglitazone up to a 55 % decrease or 1.3-fold increase in affinity was noted. Positive allosteric effects were seen by these drugs with the tamoxifen site of HSA, and neither drug had any notable binding at the digitoxin site for the normal or modified forms of HSA. Rosiglitazone also had weak interactions at a site in subdomain IB, which increased in affinity by up to 5.0-fold with the Go- or MGo-modified HSA. This study illustrated how affinity microcolumns can be used to provide a detailed analysis of solute-protein systems that involve complex interactions. The data obtained should also be valuable in providing a better understanding of how drug interactions with HSA and other proteins can be altered by modifications of these binding agents in diseases such as diabetes.

High frequency of silent mutations in gyrA gene of Mycobacterium tuberculosis in Indian isolates.

Reporting any uncommon or untapped changes in bacterial genetics or physiology would be of great importance to support the drug development process. We studied 120 Mycobacterium tuberculosis clinical isolates with different geographical origin within India and their resistance profile and found a significant number of isolates (109) harboring the polymorphism at nucleotide positions 61 and 284 of the gyrA gene. Bioinformatics analysis of these changes for drug binding suggested no significant change in the binding of the drug but have lower binding energies as compared with the wild-type proteins. Although functionally silent for the gyrA gene, these changes are indicating a silent geographical and evolutionary change that needs to be further studied for drug discovery and bacterial fitness.

Bioefficacy, chromatographic profiling and drug-likeness analysis of flavonoids and terpenoids as potential inhibitors of H1N1 influenza viral proteins

Considering medicinal plants, natural products present in these plants are the best sources of medications for combating viral infection. The possible drug target against viral H1N1 influenza proteins lead to identification of selected secondary metabolites from potential plants Tinospora cordifolia, Ocimum sanctum, and Piper nigrum. On analysis of in vitro cell based antiviral activity of the selected plant extracts, an indication for a possible lead compound against neuraminidase activity was evident. Potent ligands were selected using drug docking and ADMET analysis, and the screened lead metabolites were ultimately identified as terpenoid (Columbin) and, flavonoid (Cubebin, and Apigenin). Among the selected ligands, the drug binding activity of Cubebin with all the 6 proteins of H1N1 influenza type A virus, HA (4r8w), NA (4qn7), M2 (3lbw), PA (4wsb), PB1 (2znl) and PB2 (3wil), was pronounced. In addition, physicochemical and pharmacokinetic parameters linked to absorption, distribution, metabolism, excretion and toxicity (ADMET) have been evaluated and corroborate with our in vitro results. Molecular dynamics modelling indicated Cubebin can be a potential phytochemical in a drug discovery pipeline for the development of neuraminidase inhibitors. Further studies can provide a possibility for an alternative therapy against Influenza viruses.

Colorimetric approach based on iron oxide magnetic molecularly imprinted nanozyme for sensitive determination of antipyrine and benzocaine: Application to environmental water samples and pharmaceutical dosage form

Nanozymes provide the required enzymatic activity however, they lack the selectivity to target a certain drug during analysis. Herein, a novel polydopamine molecularly imprinted polymer (MIP) was fabricated onto Fe3O4 nanozymes surface for sensitive and selective determination of antipyrine (ANT) and benzocaine HCl (BEN). Synthesized Fe3O4 nanozyme with peroxidase like activity catalyzed the oxidation of ortho phenylenediamine by the aid of H2O2 to produce intense yellow color that can be measured via colorimetric assay. The MIPs were prepared and characterized using field-emission scanning electronic microscopy, Fourier-transform IR spectroscopy, surface area analysis, and X-ray diffraction spectrum. Two Fe3O4 @ MIP NPs nanozyme with peroxidase like activity of two drugs were prepared. Upon binding of the target drug with its respective MIP, an inhibition in the Fe3O4 enzymatic activity occurred and thus a decrease in the yellow color produced. This quantitative decrease in the color intensity represents the drug concentration. Factors affecting the enzymatic colorimetric reaction have been investigated and optimized. The formed MIPs increased the sensitivity of the proposed method with LOD 1.405, 0.658 ng mL−1, LOQ 4.259, 1.993 and recovery percentage of 100.07, 100.57 for ANT and BEN, respectively. The reusability of the Fe3O4 @ MIP NPs was assessed for six cycles without significant loss in enzymatic oxidase activity. Furthermore, the method was validated following ICH guidelines. Then, it was successfully applied for determination of the two drugs in spiked environmental water samples and pharmaceutical formulation.

Investigation the binding and partition properties of azithromycin drug using drug-surfactant mixed micelles in the presence of trisodium citrate electrolyte

The significance of mixed micelle in pharmaceutical industries is improving the binding and solubility of poorly water-soluble drugs. This work highlighted the effect of organic counterion (NAC) for improving the micellization association of zwitterionic (CAPB) and cationic drug (DPC) by surface tension measurements. The Corrin–Harkins (CH) approach assessed the CAPB, DPC and CAPB-DPC counterion binding values for DPC in the aqueous and NAC media at 25 °C. The counterion binding B for CAPB-DPC from αDPC 1.0–0.0 was found 0.536, 0.3801, 0.368, 0.355, 0.310 and 0.272 in the presence of NAC which indicated that DPC improved the counterion binding in the mixed micellar system. The Gibb’s free energy of micellization (ΔGmic°) were increased and became more negative, suggesting that the CAPB–DPC interaction was controlled by electrostatic interaction. Furthermore, the application of mixed micelle was utilized to enhance the binding constant (LogKb) and partition coefficient (LogKx) of the poorly water-soluble drug (AZI) in aqueous and NAC media at 25 °C using a UV spectrophotometer. From UV spectra, The LogKb and LogKx of AZI were lower in single micellar systems and higher in mixed micellar systems of CAPB-DPC. The maximum LogKb values at αDPC 0.4 were found 3.091, 3.233, 3.417, and 3.368 and LogKx values were found 4.749, 4.919, 5.087 and 5.049 in the presence of 0.0, 0.5, 3.0 and 20 mmol L−1 of NAC. AZI molecules are found in the palisade layer of mixed micelle when the DPC mole fraction is less than αDPC 0.6, while more than αDPC 0.6 causes steric hindrance, increasing hydrophobicity in the system. Furthermore, surfactant-drug mixed micelles could serve as poorly drug-solubilizing agents, as well as a multidrug delivery system in pharmaceutical applications.

Elucidating the binding dynamics and structural impacts of Pt(II) and Pd(II) complexes on human serum albumin: A comprehensive spectroscopic and computational study

The binding of drugs to plasma proteins, such as human serum albumin (HSA), encompasses two main functions: depot and transport, which are performed by these proteins. This binding has significant pharmacodynamic and pharmacokinetic consequences. 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline 1,2-diamino cyclohexane platinum (Pt(II)) or palladium (Pd(II)) complexes, have a similar structure. These new synthetic complexes have desirable anticancer activity, and the binding of these complexes with HSA was investigated. Spectroscopic and thermodynamic analyses indicate that the interactions between HSA and the complexes occur spontaneously with the static-dynamic quenching mechanism. However, the interactions are primarily driven by noncovalent forces such as hydrophobic interactions and π–π stacking, as evidenced by the positive enthalpy and entropy values associated with these complexes. In the circular dichroism (CD) results, HSA had fewer structural changes with the addition of the Pt(II) than the Pd(II) complex. Docking studies were performed at three Sudlow sites, I, II, and III, to identify the complex’s binding site on HSA. The reactive sites of HSA to Pt(II) and Pd(II) complexes mainly reside within its hydrophobic cavity in domain II (site II). Furthermore, molecular dynamics simulations showed low RMSD, Rg, and binding energy values during the 400 ns for the Pt(II) complex. The results suggest that the connection between the Pt(II) complex and HSA is reversible. The Pt(II) complex was a more desirable candidate than the Pd(II) complex. The results of the experimental methods are consistent with those of dynamic simulation and molecular docking methods, indicating that HSA is more stable in the presence of the Pt(II) complex.

Drug Binding to Partially Unfolded Serum Albumin: Insights into Nonsteroidal Anti-Inflammatory Drug Naproxen-BSA Interactions from Spectroscopic and MD Simulation Studies.

Understanding the binding details of a small-molecule drug to a protein in its partially unfolded state is important for drug delivery as it provides insight into the overall drug-binding ability of the protein, even when the majority of binding pockets in its unfolded state are impaired. The interaction of partially unfolded proteins with drugs remains poorly understood due to a lack of structural information on proteins in their partially unfolded states. Here, we studied the interaction between serum albumin (bovine serum albumin (BSA) as a model system), an abundant protein in blood serum that is an effective carrier for numerous known drugs, and a nonsteroidal anti-inflammatory drug (NSAID) naproxen (NPS) using various spectroscopic and computational methods. Molecular dynamics simulations starting from the drug-unbound state and performed at physiological and higher temperatures revealed novel hydrophobic sites on the BSA surface. We analyzed the BSA-NPS interaction in the presence and absence of the cationic organized assembly CTAB and two oligosaccharides (β-CD and 2-HP-β-CD) at different excitation wavelengths. The solvation dynamics of BSA under NPS-bound conditions became ∼4.6% faster. Oligosaccharides were found to increase the solubility of NPS by providing a hydrophobic environment for the formation of inclusion complexes through host-guest interactions. These findings provide a comprehensive overview and uncover the binding model and mechanism of interaction of NPS with BSA, revealing hydrophobic and electrostatic interactions and hydrogen bonds required for BSA to bind NPS at these noncanonical sites. The molecular-level understanding of the binding mechanism of commonly used NSAIDs like NPS with partially unfolded BSA will be useful in designing pharmaceutically important molecules with efficient loading and delivery properties.

Low-Basicity 5-HT6 Receptor Ligands from the Group of Cyclic Arylguanidine Derivatives and Their Antiproliferative Activity Evaluation

The serotonin 5-HT6 receptor (5-HT6R), expressed almost exclusively in the brain, affects the Cdk5 signaling as well as the mTOR pathway. Due to the association of 5-HT6R signaling with pathways involved in cancer progression, we decided to check the usefulness of 5-HT6R ligands in the treatment of CNS tumors. For this purpose, a new group of low-base 5-HT6R ligands was developed, belonging to arylsulfonamide derivatives of cyclic arylguanidines. The selected group of molecules was also tested for their antiproliferative activity on astrocytoma (1321N1) and glioblastoma (U87MG, LN-229, U-251) cell lines. Some of the molecules were subjected to ADMET tests in vitro, including lipophilicity, drug binding to plasma proteins, affinity for phospholipids, drug–drug interaction (DDI), the penetration of the membrane (PAMPA), metabolic stability, and hepatotoxicity as well as in vivo cardiotoxicity in the Danio Rerio model. Two antagonists with an affinity constant Ki < 50 nM (PR 68 Ki = 37 nM) were selected. These compounds were characterized by very high selectivity. An analysis of pharmacokinetic parameters for the lead compound PR 68 confirmed favorable properties for administration, including passive diffusion and acceptable metabolic stability (metabolized in 49%, MLMs). The compound did not exhibit the potential for drug–drug interactions.

Computationally accelerated identification of P-glycoprotein inhibitors.

Overexpression of the polyspecific efflux transporter, P-glycoprotein (P-gp, MDR1, ABCB1 ), is a major mechanism by which cancer cells acquire multidrug resistance (MDR), the resistance to diverse chemotherapeutic drugs. Inhibiting drug transport by P-gp can resensitize cancer cells to chemotherapy, but there are no P-gp inhibitors available to patients. Clinically unsuccessful P-gp inhibitors tend to bind at the pump's transmembrane drug binding domains and are often P-gp transport substrates, resulting in lowered intracellular concentration of the drug and altered pharmacokinetics. In prior work, we used computationally accelerated drug discovery to identify novel P-gp inhibitors that target the pump's cytoplasmic nucleotide binding domains. Our first-draft study provided conclusive evidence that the nucleotide binding domains of P-gp are viable targets for drug discovery. Here we develop an enhanced, computationally accelerated drug discovery pipeline that expands upon our prior work by iteratively screening compounds against multiple conformations of P-gp with molecular docking. Targeted molecular dynamics simulations with our homology model of human P-gp were used to generate docking receptors in conformations mimicking a putative drug transport cycle. We offset the increased computational complexity using custom Tanimoto chemical datasets, which maximize the chemical diversity of ligands screened by docking. Using our expanded, virtual-assisted pipeline, we identified nine novel P-gp inhibitors that reverse MDR in two types of P-gp overexpressing human cancer cell lines, reflecting a 13.4% hit rate. Of these inhibitors, all were non-toxic to non-cancerous human cells, and six were not likely to be transport substrates of P-gp. Our novel P-gp inhibitors are chemically diverse and are good candidates for lead optimization. Our results demonstrate that the nucleotide binding domains of P-gp are an underappreciated target in the effort to reverse P-gp-mediated multidrug resistance in cancer.

Structural basis for ryanodine receptor type 2 leak in heart failure and arrhythmogenic disorders

Heart failure, the leading cause of mortality and morbidity in the developed world, is characterized by cardiac ryanodine receptor 2 channels that are hyperphosphorylated, oxidized, and depleted of the stabilizing subunit calstabin-2. This results in a diastolic sarcoplasmic reticulum Ca2+ leak that impairs cardiac contractility and triggers arrhythmias. Genetic mutations in ryanodine receptor 2 can also cause Ca2+ leak, leading to arrhythmias and sudden cardiac death. Here, we solved the cryogenic electron microscopy structures of ryanodine receptor 2 variants linked either to heart failure or inherited sudden cardiac death. All are in the primed state, part way between closed and open. Binding of Rycal drugs to ryanodine receptor 2 channels reverts the primed state back towards the closed state, decreasing Ca2+ leak, improving cardiac function, and preventing arrhythmias. We propose a structural-physiological mechanism whereby the ryanodine receptor 2 channel primed state underlies the arrhythmias in heart failure and arrhythmogenic disorders.

Cell Membrane-Camouflaged Biomimetic Magnetic Fluorescent Nanoparticles with Enhanced Stability for High-Performance Lead Drug Discovery.

Biomimetic nanoengineering empowers nanoparticles with enhanced biointerfacial capabilities by directly utilizing cell membranes (CMs) of natural origin. This top-down technique provides a powerful tool for the screening of potentially active compounds in complex matrices. Herein, cartilaginous end plate (CEP) cell membrane biomimetic Nile red (NR)-loaded zeolitic imidazolate frameworks-8 (ZIF-8) modified magnetic graphene oxide (CEP/MGO-ZIF-8-NR) nanocomposites with enhanced stability were accurately prepared by chemical bonding and used as a drug discovery platform for the specific identification and effective extraction of drug leads with anti-intervertebral disc degeneration (IDD) in Yaobitong capsules (YBTCs). The constructed CEP/MGO-ZIF-8-NR exhibited excellent magnetic properties, fluorescence properties, and stability. In addition, drug binding experiments showed that the CEP/MGO-ZIF-8-NR nanocomposites possessed higher adsorption capacity, faster adsorption rate, and superior selectivity compared with uncoated MGO-ZIF-8-NR. Ultimately, four potential bioactive molecules, including ginsenoside Ro, ginsenoside Rg1, astringin, and chikusetsusaponin V methyl ester, were successfully screened and identified in vitro from YBTC. The results of the CCK-8 assay and BrdU ELISA kit showed that the screened compounds promoted CEP cell proliferation in a concentration-dependent manner. Cellular distribution experiments revealed that CEP/MGO-ZIF-8-NR could rapidly escape from lysosomes and into the cytoplasm. And the pharmacological activity of these compounds was further confirmed by real-time cytomorphological imaging of CEP cells by confocal laser scanning microscopy (CLSM). Overall, this surface engineering strategy endows bioaffinity sample pretreatment materials with tremendous versatility, improves drug screening efficiency, and broadens the horizons and methodologies for drug lead discovery.

Integration of Hydrogen-Deuterium Exchange Mass Spectrometry with Molecular Dynamics Simulations and Ensemble Reweighting Enables High Resolution Protein-Ligand Modeling.

Hydrogen-Deuterium exchange mass spectrometry's (HDX-MS) utility in identifying and characterizing protein-small molecule interaction sites has been established. The regions that are seen to be protected from exchange upon ligand binding indicate regions that may be interacting with the ligand, giving a qualitative understanding of the ligand binding pocket. However, quantitatively deriving an accurate high-resolution structure of the protein-ligand complex from the HDX-MS data remains a challenge, often limiting its use in applications such as small molecule drug design. Recent efforts have focused on the development of methods to quantitatively model Hydrogen-Deuterium exchange (HDX) data from computationally modeled structures to garner atomic level insights from peptide-level resolution HDX-MS. One such method, HDX ensemble reweighting (HDXer), employs maximum entropy reweighting of simulated HDX data to experimental HDX-MS to model structural ensembles. In this study, we implement and validate a workflow which quantitatively leverages HDX-MS data to accurately model protein-small molecule ligand interactions. To that end, we employ a strategy combining computational protein-ligand docking, molecular dynamics simulations, HDXer, and dimensional reduction and clustering approaches to extract high-resolution drug binding poses that most accurately conform with HDX-MS data. We apply this workflow to model the interaction of ERK2 and FosA with small molecule compounds and inhibitors they are known to bind. In five out of six of the protein-ligand pairs tested, the HDX derived protein-ligand complexes result in a ligand root-mean-square deviation (RMSD) within 2.5 Å of the known crystal structure ligand.

NTRK Fusion as an Acquired Mechanism of Resistance to Selpercatinib in RET Positive Thyroid Cancer

Background: Rearrangement during Transfection (RET) proto-oncogene is involved in the pathogenesis of various thyroid cancer subtypes and drugs that block the RET tyrosine kinase pathways are used in the management of locally advanced and metastatic Medullary Thyroid Cancer (MTC). Acquired resistance to RET inhibitors likely occurs via two different mechanisms: (1) On-target mutations that prevent drug binding and (2) Activated alternative mechanisms which bypass the targeted kinase. We present a unique mechanism of selpercatinib resistance via secondary NTRK fusion mutation in a case of RET altered medullary thyroid cancer. Case Presentation: The patient is a 72-year-old female with stage III metastatic MTC who developed recurrence with newly found RET mutation 7 years after complete surgical resection. Despite initial stable response with selpercatinib for 2 years she rapidly progressed with widespread disease. Cell-free DNA (cfDNA) testing done at the time of progression revealed an NTRK1 fusion. Unfortunately, the patient succumbed to the disease despite starting targeted therapy with larotrectinib. Conclusion: This case highlights the importance of further investigation into mechanisms of resistance to RET inhibitors and drug studies directed towards overcoming them