High Affinity Binding Research Articles

Rapid Nuclease-Assisted Selection of High-Affinity Small-Molecule Aptamers.

Aptamers are nucleic acid bioreceptors that have been widely utilized for a variety of biosensing applications, including in vivo detection methods that would not be possible with antibody-based systems. However, it remains challenging to generate high-quality aptamers for small molecule targets, particularly for use under physiological conditions. We present a highly effective aptamer selection technology for small-molecule targets that utilizes the nuclease EcoRI to remove nonspecific or weakly binding sequences in solution phase, rapidly enriching high-affinity target binders within just a few rounds of selection. As proof-of-concept, we used our nuclease-assisted SELEX (NA-SELEX) method to isolate aptamers for a synthetic cannabinoid, AB-FUBINACA. Within five rounds, we identified two highly specific aptamers that exhibit nanomolar affinity at physiological temperature. We also demonstrate the robustness and reproducibility of NA-SELEX by performing the same selection experiment with fresh reagents and libraries, obtaining the same two aptamers as well as two other high-quality aptamer candidates. Finally, we compare NA-SELEX against a conventional library-immobilized SELEX screen for AB-FUBINACA using the same screening conditions, identifying aptamers with 25-100-fold weaker affinity after 11 rounds of selection. NA-SELEX therefore could be an effective selection method for the isolation of high-quality aptamers for small-molecule targets.

Molecular mechanisms of proteoglycan-mediated semaphorin signaling in axon guidance

The precise assembly of a functional nervous system relies on axon guidance cues. Beyond engaging their cognate receptors and initiating signaling cascades that modulate cytoskeletal dynamics, guidance cues also bind components of the extracellular matrix, notably proteoglycans, yet the role and mechanisms of these interactions remain poorly understood. We found that Drosophila secreted semaphorins bind specifically to glycosaminoglycan (GAG) chains of proteoglycans, showing a preference based on the degree of sulfation. Structural analysis of Sema2b unveiled multiple GAG-binding sites positioned outside canonical plexin-binding site, with the highest affinity binding site located at the C-terminal tail, characterized by a lysine-rich helical arrangement that appears to be conserved across secreted semaphorins. In vivo studies revealed a crucial role of the Sema2b C-terminal tail in specifying the trajectory of olfactory receptor neurons. We propose that secreted semaphorins tether to the cell surface through interactions with GAG chains of proteoglycans, facilitating their presentation to cognate receptors on passing axons.

PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade

Programmed cell death ligand 2 (PD-L2), a ligand for the receptor programmed cell death 1 (PD-1), has an identity of 34% with its twin ligand PD-L1 and exhibits higher binding affinity with PD-1 than PD-L1. However, the role of PD-L2 in non-small cell lung cancer (NSCLC) progression, especially tobacco-induced cancer progression, has not been fully understood. Here, we found that PD-L2 promoted tumor growth in murine models with recruitment of regulatory T cells (Tregs). In patients with NSCLC, PD-L2 expression level in tumor samples was higher than in counterpart normal controls and was positively associated with patients’ response to anti-PD-1 treatment. Mechanismly, PD-L2 bound its receptor Repulsive guidance molecule B (RGMB) on cancer cells and activated extracellular signal-regulated kinase (Erk) and nuclear factor κB (NFκB), leading to increased production of chemokine CCL20, which recruited Tregs and contributed to NSCLC progression. Consistently, knockdown of RGMB or NFκB p65 inhibited PD-L2-induced CCL20 production, and silencing of PD-L2 repressed Treg recruitment by NSCLC cells. Furthermore, cigarette smoke and carcinogen benzo(a)pyrene (BaP) upregulated PD-L2 in lung epithelial cells via aryl hydrocarbon receptor (AhR)-mediated transcription activation, whose deficiency markedly suppressed BaP-induced PD-L2 upregulation. These results suggest that PD-L2 mediates tobacco-induced recruitment of Tregs via the RGMB/NFκB/CCL20 cascade, and targeting this pathway might have therapeutic potentials in NSCLC.

Molecular Regulator Driving Endometriosis Towards Endometrial Cancer: A Multi-Scale Computational Investigation to Repurpose Anti-Cancer drugs.

Endometriosis is a gynecological disorder among reproductive-aged women. Recent epidemiological investigations suggest endometriosis increases the risk of endometrial cancer. However, the molecular entity leading to endometriosis-to-endometrial cancer is largely unknown. This study aimed to combine a variety of computational approaches to identify the key therapeutic target promoting endometriosis-to-endometrial cancer and screen potential inhibitors against target to prevent cancer development. Our systematic investigations, includes transcriptomic profiling, protein network, pharmacophore modeling, docking, binding free energy calculation, dynamics simulation, and quantum mechanics. The gene expression analysis on endometriosis and endometrial cancer was performed and showed 108 shared upregulated genes in both conditions. Further construction of interaction network with 108 genes showed intercellular adhesion molecule 1 (ICAM1) to be a crucial molecule with a high degree of connectivity that influences vital mechanisms related to cancer pathways. We then generated ligand-based pharmacophore models using established ICAM1 inhibitors. Among the models, the ADRRR_8 pharmacophore exhibited a robust area under curve (AUC = 0.83), was employed to screen 1739 anti-cancer drugs. On screening, 421 anti-cancer drugs displayed ICAM1-inhibiting pharmacophore features. Further, the docking of 421 drugs with ICAM1 showed lanreotide (-7.80 kcal/mol) with better affinity than the reference ICAM1 inhibitor (-3.59 kcal/mol). Further validation though binding free energy and dynamics simulation of the lanreotide-ICAM1 complex showed a high binding affinity of -55.90 kcal/mol and contributed stable confirmation. According to quantum chemical calculations, lanreotide's electronic properties favour ICAM1 binding with highest occupied molecular orbital was -6.91 eV and lowest unoccupied molecular orbital was -3.93 eV. Our study supports using lanreotide to treat endometriosis, which could delay or prevent endometrial cancer. These predictions need to be confirmed and examined to determine the use of lanreotide in endometriosis treatment.

Structure based functional identification of an uncharacterized protein from Coxiella burnetii involved in adipogenesis

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen posing a significant global public health threat. There is a pressing need for dependable and effective treatments, alongside an urgency for further research into the molecular characterization of its genome. Within the genomic landscape of Coxiella burnetii, numerous hypothetical proteins remain unidentified, underscoring the necessity for in-depth study. In this study, we conducted comprehensive in silico analyses to identify and prioritize potential hypothetical protein of Coxiella burnetii, aiming to elucidate the structure and function of uncharacterized protein. Furthermore, we delved into the physicochemical properties, localization, and molecular dynamics and simulations, and assessed the primary, secondary, and tertiary structures employing a variety of bioinformatics tools. The in-silico analysis revealed that the uncharacterized protein contains a conserved Mth938-like domain, suggesting a role in preadipocyte differentiation and adipogenesis. Subcellular localization predictions indicated its presence in the cytoplasm, implicating a significant role in cellular processes. Virtual screening identified ligands with high binding affinities, suggesting the protein’s potential as a drug target against Q fever. Molecular dynamics simulations confirmed the stability of these complexes, indicating their therapeutic relevance. The findings provide a structural and functional overview of an uncharacterized protein from C. burnetii, implicating it in adipogenesis. This study underscores the power of in-silico approaches in uncovering the biological roles of uncharacterized proteins and facilitating the discovery of new therapeutic strategies. The findings provide valuable preliminary data for further investigation into the protein’s role in adipogenesis.

Virtual screening and molecular dynamics simulation of natural compounds as potential inhibitors of serine/threonine kinase 16 for anticancer drug discovery.

Serine/threonine kinase 16 (STK 16) is involved in many facets of cellular regulation; activation of STK 16 plays a crucial role in the migration of cancer cells. Therefore, it is a novel target for the discovery of anticancer agents. Herein, virtual screening and dynamics simulation were used to screen a large library of natural compounds against STK 16 using Schrodinger suit 2021-2 and GROMACS 2021.6. The results predicted five molecules with high binding affinity against the target, with NPC132329 (Arcyriaflavin C) and NPC160898 having higher binding affinity and molecular mechanics generalized born surface area (MM/GBSA), suggesting that it is better than the standard inhibitor. The molecular dymanics (MD) simulation studies showed that the STK 16-NPC132329 complex has the lowest root mean square deviation, and STK 16-NPC160898 was the most stable compared with the standard drug and selective STK 16 inhibitor. The minimal fluctuation was observed in the STK 16-NPC132329 and STK 16-NPC160898 complexes based on the root mean square fluctuation trajectory with NPC132329 and NPC160898 forming 2 and 3 hydrogen bonds respectively with the amino acid residue of the target's binding site. Overall, NPC132329 and NPC160898 are better STK 16 inhibitors than the standard drug and selective inhibitor, which can be further studied to discover novel anticancer drugs.

An In-Silico Drug Designing Approach Attempted on a Newly Synthesized Co(II) Complex along with its Other Biological Activities: A Combined Investigation of both Experimental and Theoretical Aspects.

A new Co (II) complex incorporating a novel Schiff base ligand acquired from the condensation of 3,3'-Methylenedianiline and 2-Hydroxy-5-bromobenzaldehyde was synthesized and characterized. The synthesized complex was air and moisture stable, monomeric, and non-electrolytic in nature. Based on physical and spectral studies, tetrahedral conformation was ascribed to the synthesized Co (II) complex.Density Functional Theory (DFT) was used to analysis different electronic parameters of the optimized structure of Co(II) complex to reveal its stability.Using different analytic and spectroscopic techniques, the new Co (II) complex was established to interact with DNA quite effectively and works as an efficient metallo intercalators. The synthesized complex was discovered to cleave DNA significantly, so it can be inferred that the complex will inhibit the growth of pathogens. Molecular docking was performed to check the binding affinity of the cobalt complex with different receptors, responsible for different diseases. Proteins like progesterone receptor and induced myeloid leukemia cell differentiation Mcl-1 protein showed high binding affinity with this complex, and hence the complex might have some implications for inhibition of progesterone hormones in biological systems. Biological activity of the Co (II) complex was also predicted through computational analysis with SwissADME.Using strains of Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus, an in vitro antibacterial activity of the ligand and Co (II) complex was carried out. This activity was further validated by a molecular docking investigation.

Argonaute protein assisted drug discovery for miRNA-181c-5p and target gene ATM translation repression: a computational approach.

The miRNA binds to AGO's seed region, prompting the exploration of small molecules that can offset miRNA repression of target mRNA. This miRNA-181c-5p was found to be upregulated in the chronic traumatic encephalopathy, a prevalent neurodegenerative disease in contact sports and military personals. The research aimed to identify compounds that disrupt the AGO-assisted loop formation between miRNA-181c-5p and ATM, consequently repressing the translation of ATM. Target genes from commonly three databases (DIANA-microT-CDS, miRDB, RNA22 and TargetScan) were subjected to functional annotation and clustering analysis using DAVID bioinformatics tool. Haddock server were employed to make miRNA-181c-5p:ATM-AGO complex. A total of 2594 small molecules were screened using Glide XP based on their highest binding affinity towards the complex, through a three-phase docking approach. The top 5 compounds (DB00674-Galantamine, DB00371-Meprobamate, DB00694-Daunorubicin, DB00837-Progabide, and DB00851-Dacarbazine) were further analyzed for stability in the miRNA-181c-5p:ATM-AGO-ligand complex interaction using GROMACS (version 2023.2). Hence, these findings suggest that these molecules hold potential for facilitating AGO-assisted repression of ATM gene translation.

Telmisartan loaded lipid nanocarrier as a potential repurposing approach to treat glioma: Characterization, apoptosis evaluation in U87MG cells, pharmacokinetic and molecular simulation study.

The study explores anticancer potential of telmisartan (TS) loaded lipid nanocarriers (TLNs) in glioma cells as a potential repurposing nanomodality along with estimation of drug availability at rat brain. Experimental TLNs were produced by previously reported method and characterized. In vitro anticancer efficacy of experimental TLNs was estimated by MTT, confocal microscopy, and FACs analysis in glioma cells. Plasma and brain pharmacokinetic (PK) parameters were also analysed by LCMS/MS. Spherical, nanosized, homogenous, unilamellar, TLNs were reported having desirable drug loading (9.5 ± 0.6 %), negative zeta potential and sustained TS release tendency. FITC-TLNs were sufficiently internalized into U87MG cells line within 0.5 h incubation period. IC50 for TLNs was considerably higher than free TS in the tested glioma cell lines. Further, TLNs induced superior apoptotic effect in U87MG cells than TS. PK (plasma/brain) data depicted higher AUC, Vss, MRT with lower Clt for TLNs suggesting improved bioavailability, in vivo residence and sustained drug availability than free TS administration. Docking studies rationalized in vitro/in vivo results as preferably higher binding affinity (docking score:12.4) was detected for TS with glioma proteins. Further, in vivo studies in glioma bearing xenograft model is underway for futuristic clinical validation of TLNs.

Hybrid Virtual Screening Approach to Predict Novel Natural Compounds against HIV-1 CCR5.

Human immunodeficiency virus (HIV) infection continues to pose a major global health challenge. HIV entry into host cells via membrane fusion mediated by the viral envelope glycoprotein gp120/gp41 is a key step in the HIV life cycle. CCR5, expressed on CD4+ T cells and macrophages, acts as a coreceptor facilitating HIV-1 entry. The CCR5 antagonist maraviroc is used to treat HIV infection. However, it can cause adverse effects and has limitations such as only inhibiting CCR5-tropic viruses. There remains a need to develop alternative CCR5 inhibitors with improved safety profiles. Natural products may offer advantages over synthetic inhibitors including higher bioavailability, binding affinity, effectiveness, lower toxicity, and molecular diversity. However, screening the vast chemical space of natural compounds to identify novel CCR5 inhibitors presents challenges. This study aimed to address this gap through a hybrid ligand-based pharmacophore modeling and molecular docking approach to virtually screen large natural product databases. A reliable pharmacophore model was developed based on 311 known CCR5 antagonists and validated against an external data set. Five natural product databases containing over 306,000 compounds were filtered based on drug-likeness rules. The validated pharmacophore model screened the databases to identify 611 hits. Key residues of the CCR5 receptor crystal structure were identified for docking. The top hits were docked, and interactions were analyzed. Molecular dynamics simulations were conducted to examine complex stability. Computational prediction evaluated pharmacokinetic properties. Three compounds exhibited similar interactions and binding energies to maraviroc. MD simulations demonstrated complex stability comparable to maraviroc. One compound showed optimal predicted absorption, minimal metabolism, and a lower likelihood of interactions than maraviroc. This computational screening workflow identified three natural compounds with promising CCR5 inhibition and favorable pharmacokinetic profiles. One compound emerged as a lead based on bioavailability potential and minimal interaction risk. These findings present opportunities for developing alternative CCR5 antagonists and warrant further experimental investigation. Overall, the hybrid virtual screening approach proved effective for mining large natural product spaces to discover novel molecular entities with drug-like properties.

Unraveling Grewia bilamellata Gagnep. Role in cerebral ischemia: Comprehensive in vivo and in silico studies.

The present study investigated the neuroprotective properties of whole plants of Grewia bilamellata Gagnep. extract (GBEE) against cerebral ischemia by harnessing both In vivo studies in a rat model and In silico studies focusing on nitric oxide synthase (NOS) inhibition. High-resolution liquid chromatography‒mass spectrometry (HR LC‒MS) analysis identified 32 phytochemicals in the GBEE, 15 of which adhered to Lipinski's rule of five. These compounds exhibited diverse physicochemical properties and high binding affinity to NOS, with cleomiscosin D showing the greatest potential. In vivo, GBEE had significant neuroprotective effects on bilateral common carotid artery occlusion/reperfusion (BCCAO/R) in rats, especially at doses of 200mg/kg and 400mg/kg body weight. GBEE treatment improved brain function, as evidenced by EEG normalization, substantial reductions in cerebral infarction size, mitigated neuronal loss, and the restoration of regular histological arrangement in the CA1 hippocampal area of the brain. Furthermore, GBEE enhanced antioxidant defenses by augmenting the activity of catalase (CAT) and superoxide dismutase (SOD), reducing malondialdehyde (MDA) levels, and restoring reduced glutathione (GSH) levels. These effects were accompanied by a decrease in nitric oxide (NO) levels, indicative of attenuated oxidative and nitrosative stress. Collectively, our findings suggest that GBEE is a promising natural therapeutic agent that may prevent or alleviate ischemic brain injury through a multifaceted mechanism involving NOS inhibition and attenuation of the oxidative stress response. This study highlights the therapeutic potential of GBEE and warrants further research into its mechanism of action and possible clinical applications.

In Silico drug repurposing pipeline using deep learning and structure based approaches in epilepsy

Due to considerable global prevalence and high recurrence rate, the pursuit of effective new medication for epilepsy treatment remains an urgent and significant challenge. Drug repurposing emerges as a cost-effective and efficient strategy to combat this disorder. This study leverages the transformer-based deep learning methods coupled with molecular binding affinity calculation to develop a novel in-silico drug repurposing pipeline for epilepsy. The number of candidate inhibitors against 24 target proteins encoded by gain-of-function genes implicated in epileptogenesis ranged from zero to several hundreds. Our pipeline has repurposed the medications with most anti-epileptic drugs and nearly half psychiatric medications, highlighting the effectiveness of our pipeline. Furthermore, Lomitapide, a cholesterol-lowering drug, first emerged as particularly noteworthy, exhibiting high binding affinity for 10 targets and verified by molecular dynamics simulation and mechanism analysis. These findings provided a novel perspective on therapeutic strategies for other central nervous system disease.

Identification and Design of Novel Potential Antimicrobial Peptides Targeting Mycobacterial Protein Kinase PknB.

Antimicrobial peptides have gradually gained advantages over small molecule inhibitors for their multifunctional effects, synthesising accessibility and target specificity. The current study aims to determine an antimicrobial peptide to inhibit PknB, a serine/threonine protein kinase (STPK), by binding efficiently at the helically oriented hinge region. A library of 5626 antimicrobial peptides from publicly available repositories has been prepared and categorised based on the length. Molecular docking using ADCP helped to find the multiple conformations of the subjected peptides. For each peptide served as input the tool outputs 100 poses of the subjected peptide. To maintain an efficient binding for relatively a longer duration, only those peptides were chosen which were seen to bind constantly to the active site of the receptor protein over all the poses observed. Each peptide had different number of constituent amino acid residues; the peptides were classified based on the length into five groups. In each group the peptide length incremented upto four residues from the initial length form. Five peptides were selected for Molecular Dynamic simulation in Gromacs based on higher binding affinity. Post-dynamic analysis and the frame comparison inferred that neither the shorter nor the longer peptide but an intermediate length of 15 mer peptide bound well to the receptor. Residual substitution to the selected peptides was performed to enhance the targeted interaction. The new complexes considered were further analysed using the Elastic Network Model (ENM) for the functional site's intrinsic dynamic movement to estimate the new peptide's role. The study sheds light on prospects that besides the length of peptides, the combination of constituent residues equally plays a pivotal role in peptide-based inhibitor generation. The study envisages the challenges of fine-tuned peptide recovery and the scope of Machine Learning (ML) and Deep Learning (DL) algorithm development. As the study was primarily meant for generation of therapeutics for Tuberculosis (TB), the peptide proposed by this study demands meticulous invitro analysis prior to clinical applications.

QSAR, ADMET, molecular docking, and dynamics studies of 1,2,4-triazine-3(2H)-one derivatives as tubulin inhibitors for breast cancer therapy

Breast cancer remains a leading cause of cancer-related deaths among women globally, necessitating the development of more effective therapeutic agents with minimal side effects. This study explores novel 1,2,4-triazine-3(2H)-one derivatives as potential inhibitors of Tubulin, a pivotal protein in cancer cell division, highlighting a targeted approach in cancer therapy. Using an integrated computational approach, we combined quantitative structure–activity relationship (QSAR) modeling, ADMET profiling, molecular docking, and molecular dynamics simulations to evaluate and predict the efficacy and stability of these compounds. Our QSAR models, developed through rigorous statistical analysis, revealed that descriptors such as absolute electronegativity and water solubility significantly influence inhibitory activity, achieving a predictive accuracy (R2) of 0.849. Molecular docking studies identified compounds with high binding affinities, particularly Pred28, which exhibited the best docking score of − 9.6 kcal/mol. Molecular dynamics simulations conducted over 100 ns provided further insights into the stability of these interactions. Pred28 demonstrated notable stability, with the lowest root mean square deviation (RMSD) of 0.29 nm and root mean square fluctuation (RMSF) values indicative of a tightly bound conformation to Tubulin. The novelty of this work lies in its methodological rigor and the integration of multiple advanced computational techniques to pinpoint compounds with promising therapeutic potential. Our findings advance the current understanding of Tubulin inhibitors and open avenues for the synthesis and experimental validation of these compounds, aiming to offer new solutions for breast cancer treatment.

The Impact of Lebrikizumab on Vaccine-Induced Immune Responses: Results from a Phase3 Study in Adult Patients with Moderate-to-Severe Atopic Dermatitis.

Lebrikizumab, a high-affinity IgG4 monoclonal antibody that selectively inhibits interleukin-13 with high binding affinity and slow dissociation rate, prevents the formation of the interleukin-4Rα/interleukin-13Rα1heterodimer receptor signaling complex. Here we report the impact of lebrikizumab on responses to two non-live vaccines in adult patients with moderate-to-severe atopic dermatitis (AD). ADopt-VA (NCT04626297) was a double-blind, placebo-controlled, parallel-group, 16-week, phase3 randomized study to assess the impact of lebrikizumab treatment on non-live vaccine immune responses, and efficacy and safety of lebrikizumab compared with placebo. Eligible patients included adults from 18 to 55years of age with moderate-to-severe chronic AD who were randomly assigned 1:1 to lebrikizumab 250mg every 2weeks or placebo and stratified according to disease severity. The primary endpoints were the development of a booster response to tetanus toxoid and a positive antibody response to meningococcal conjugate vaccine (MCV), 4weeks after administration of the corresponding vaccine. At week16, 73.6% of patients in the lebrikizumab group (n = 78/106) achieved Tdap booster response compared with 73.4% of patients in the placebo group (n = 58/79). MCV vaccine response was observed in 86.9% of patients in the lebrikizumab group (n = 86/99) and 75.0% of patients in the placebo group (n = 60/80). At week16, IGA0,1 with ≥ 2-point improvement from baseline was achieved by 40.6% (n = 51/125) of patients treated with lebrikizumab and 18.9% (n = 23/122) of patients who received placebo (p < 0.001). There was a higher proportion of patients achieving EASI75 at week16 in the lebrikizumab-treated patients (58.0%, n = 72/125) compared with placebo (32.7%, n = 40/122, p < 0.001). Treatment with lebrikizumab did not impact response to non-live vaccines Tdap and MCV in this study. Lebrikizumab treatment had a significant degree of efficacy compared to placebo across multiple endpoints. ClinicalTrials.gov identifier NCT04626297.

Superior cuproptotic efficacy of diethyldithiocarbamate-Cu4O3 nanoparticles over diethyldithiocarbamate-Cu2O nanoparticles in metastatic hepatocellular carcinoma

Metastatic hepatocellular carcinoma (HC) is a serious health concern. The stemness of cancer stem cells (CSCs) is a key driver for HC tumorigenesis, apoptotic resistance, and metastasis, and functional mitochondria are critical for its maintenance. Cuproptosis is Cu-dependent non-apoptotic pathway (mitochondrial dysfunction) via inactivating mitochondrial enzymes (pyruvate dehydrogenase “PDH” and succinate dehydrogenase “SDH”). To effectively treat metastatic HC, it is necessary to induce selective cuproptosis (for halting cancer stemness genes) with selective oxidative imbalance (for increasing cell susceptibility to cuproptosis and inducing non-CSCs death). Herein, two types of Cu oxide nanoparticles (Cu4O3 “C(I + II)” NPs and Cu2O “C(I)” NPs) were used in combination with diethyldithiocarbamate (DD, an aldehyde dehydrogenase “ALDH” inhibitor) for comparative anti-HC investigation. DC(I + II) NPs exhibited higher cytotoxicity, mitochondrial membrane potential, and anti-migration impact than DC(I) NPs in the treated human HC cells (HepG2 and/or Huh7). Moreover, DC(I + II) NPs were more effective than DC(I) NPs in the treatment of HC mouse groups. This was mediated via higher selective accumulation of DC(I + II) NPs in only tumor tissues and oxidant activity, causing stronger selective inhibition of mitochondrial enzymes (PDH, SDH, and ALDH2) than DC(I)NPs. This effect resulted in more suppression of tumor and metastasis markers as well as stemness gene expressions in DC(I + II) NPs-treated HC mice. In addition, both nanocomplexes normalized liver function and hematological parameters. The computational analysis found that DC(I + II) showed higher binding affinity to most of the tested enzymes. Accordingly, DC(I + II) NPs represent a highly effective therapeutic formulation compared to DC(I) NPs for metastatic HC.

Identification of a Novel Protein Phosphatase 2A Activator, PPA24, as a Potential Therapeutic for FOLFOX-Resistant Colorectal Cancer.

A series of compounds were designed utilizing molecular modeling and fragment-based design based upon the known protein phosphatase 2A (PP2A) activators, NSC49L and iHAP1, and evaluated for their ability to inhibit the viability of colorectal cancer (CRC) and folinic acid, 5-fluorouracil, and oxaliplatin (FOLFOX)-resistant CRC cells. PPA24 (19a) was identified as the most cytotoxic compound with IC50 values in the range of 2.36-6.75 μM in CRC and FOLFOX-resistant CRC cell lines. It stimulated PP2A activity to a greater extent, displayed lower binding energies through molecular docking, and showed higher binding affinity through surface plasmon resonance for PP2A catalytic subunit α than the known PP2A activators. PPA24 dose-dependently induced apoptosis and oxidative stress, decreased the level of c-Myc expression, and synergistically potentiated cytotoxicity when combined with gemcitabine and cisplatin. Furthermore, a PPA24-encapsulated nanoformulation significantly inhibited the growth of CRC xenografts without systemic toxicities. Together, these results signify the potential of PPA24 as a novel PP2A activator and a prospective therapeutic for CRC and FOLFOX-resistant CRC.

Designing multi-epitope vaccine against human cytomegalovirus integrating pan-genome and reverse vaccinology pipelines

Human cytomegalovirus (HCMV) is accountable for high morbidity in neonates and immunosuppressed individuals. Due to the high genetic variability of HCMV, current prophylactic measures are insufficient. In this study, we employed a pan-genome and reverse vaccinology approach to screen the target for efficient vaccine candidates. Four proteins, envelope glycoprotein M, UL41A, US23, and US28, were shortlisted based on cellular localization, high solubility, antigenicity, and immunogenicity. A total of 29 B-cell and 44 T-cell highly immunogenic and antigenic epitopes with high global population coverage were finalized using immunoinformatics tools and algorithms. Further, the epitopes that were overlapping among the finalized B-cell and T-cell epitopes were linked with suitable linkers to form various combinations of multi-epitopic vaccine constructs. Among 16 vaccine constructs, Vc12 was selected based on physicochemical and structural properties. The docking and molecular simulations of VC12 were performed, which showed its high binding affinity (−23.35 kcal/mol) towards TLR4 due to intermolecular hydrogen bonds, salt bridges, and hydrophobic interactions, and there were only minimal fluctuations. Furthermore, Vc12 eliciting a good response was checked for its expression in Escherichia coli through in silico cloning and codon optimization, suggesting it to be a potent vaccine candidate.

Differential effects of the N-terminal helix of FGF8b on the activity of a small-molecule FGFR inhibitor in cell culture and for the extracellular domain of FGFR3c in solution.

SSR128129E (SSR) is a unique small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high-affinity allosteric binder that selectively blocks one of the two major FGFR-mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain. The binding site overlaps with the position of an N-terminal helix, an element exclusive for the FGF8b growth factor, which could potentially convert SSR from an allosteric inhibitor into an orthosteric blocker for the particular FGFR/FGF8b system. In this regard, we report here on the structural and functional investigation of FGF8b/FGFR3c system and the effects imposed on it by SSR. We show that SSR is equally or more potent in inhibiting FGF8b-induced FGFR signaling compared to FGF2-induced activation. On the other hand, when studied in the context of separate extracellular domains of FGFR3c in solution with NMR spectroscopy, SSR is unable to displace the N-terminal helix of FGF8b from its binding site on FGFR3c and behaves as a weak orthosteric inhibitor. The substantial inconsistency between the results obtained with cell culture and for the individual water-soluble subdomains of the FGFR proteins points to the important role played by the cell membrane.

Ligand-Based Virtual Screening for Discovery of Indole Derivatives as Potent DNA Gyrase ATPase Inhibitors Active against Mycobacterium tuberculosis and Hit Validation by Biological Assays.

Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 μM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 μg/mL, respectively, superior to novobiocin (MIC > 100.0 μg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.