Tertiary Structure Research Articles

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.

PD1+CD4+ T cells promote receptor editing and suppress autoreactivity of CD19+CD21low B cells within the lower respiratory airways in adenovirus pneumonia

Human adenovirus (HAdV) pneumonia poses a major health burden for young children, however, factors that contribute to disease severity remain elusive. We analyzed immune cells from bronchoalveolar lavage (BAL) of children with HAdV pneumonia and found that CD19+CD21low B cells were significantly enriched in the BAL and were associated with increased autoantibody concentrations and disease severity. Myeloid cells, PD-1+CD4+ T helper cells and CD21low B cells formed tertiary lymphoid structures within the respiratory tracts. Myeloid cells promoted autoantibody production by expressing high amounts of B cell activating factor (BAFF). In contrast, PD-1+CD4+ T helper cells induced production of IgG1 and IgG3 antibodies but suppressed autoreactive IgGs by initiating B cell receptor editing. In summary, this study reveals cellular components involved in protective versus autoreactive immune pathways in the respiratory tract, and these findings provide potential therapeutic targets for severe HAdV lower respiratory tract infections.

Unexpected diselenide metathesis in selenocysteine-substituted biologically active peptides.

Substitution of disulfide bonds with a diselenide bonds in peptides and proteins is an often-used strategy to increase the stability of naturally occurring peptides and proteins. In this paper, diselenide metathesis between model diselenide dimer peptides, as well as that in diselenide(s)-substituted biologically active peptides, were analyzed. Surprisingly, depending on the tertiary structure of the peptides, we observed that the metathesis reaction occurs under physiological conditions even in the absence of reducing agents, light and heating.

Analysis of Immunological Biomarkers Associated With Rejection After Uterus Transplantation in Human.

Uterus transplantation (UTx) is an emerging therapy for women with uterine infertility. However, critical questions remain with this procedure including the mechanisms involved in graft rejection. In this study, we analyzed the immune profile of ectocervical biopsies from 5 patients after UTx before and during their first episode of rejection using RNA sequencing, quantitative polymerase chain reaction, and imaging mass cytometry. We identified 530 upregulated and 207 downregulated genes associated with graft rejection. Enrichment databases revealed abnormalities of skin-associated genes and the immune system, in particular activation of T and B lymphocytes, and macrophages. Imaging mass cytometry confirmed these observations; in cervical biopsies of 3 women, rejection was associated with the presence of B-cell structures linked to tertiary lymphoid structures, and 2 biopsies from 1 woman with severe rejection episodes and poor prognosis of graft function (repeated miscarriage and implantation failures) were associated with an accumulation of HLA-DR- macrophages, producing granzyme B at the surface of the epithelium. We showed that rejection of a UTx graft was associated with major alterations of immune markers including the involvement of tertiary lymphoid structures, the most organized of which may be a sign of chronic rejection, and with an increase in HLA-DR- macrophages expressing granzyme B in the case of grade 3 rejection episodes according Mölne's classification. We identified potential emerging biomarkers to predict or diagnose graft rejection (Keratin 1 granzyme B, IL1β). These findings could lead to development of improved strategies for the identification, prevention, and/or treatment of uterus graft rejection.

Effect of dry- and moist-heat treatment processes on the structure, solubility, and in vitro digestion of macadamia protein isolate.

This study aimed to enhance the solubility and digestibility of macadamia protein isolate (MPI) for potential utilization in the food industry. The impact of dry- and moist-heat treatments at various temperatures (80, 90, and 100°C) and durations (15 and 30min) on macadamia protein's microstructure, solubility, molecular weight, secondary and tertiary structure, thermal stability, and digestibility were investigated and evaluated. The heating degree was found to cause roughening of the MPI surface. The solubility of MPI after dry-heat treatment for 15 min at 100°C reached 290.96±2.80% relative to that of untreated protein. Following heat treatment, the bands of protein macromolecules disappeared, while MPI was stretched by vibrations of free and hydrogen-bonded hydroxyl groups. Additionally, an increase in thermal stability was observed. After heat treatment, hydrophobic groups inside the protein are exposed. Heat treatment significantly improved the in vitro digestibility of MPI, reaching twice that of untreated protein. The results also demonstrated that dry- and moist-heat treatments have distinct impacts on MPI, while heating temperature and duration affect the degree of modification. With a decreased ordered structure and increased random coil content, the dry-heat treatment significantly enhanced the in vitro digestibility of MPI. The digestibility of MPI after dry-heat treatment for 30 min at 90°C increased by 77.82±2.80% compared to untreated protein. Consequently, compared to moist-heat treatment, dry-heat treatment was more effective in modifying macadamia protein. Dry-heat treatment of 30 min at 90°C was determined as the optimal condition. PRACTICAL APPLICATION: Heat treatment enhances MPI characteristics, potentially advancing macadamia-derived food production, including plant-based beverages and protein supplements.

CNRBind: Small Molecule-RNA Binding Sites Recognition via Site Significant from Nucleotide and Complex Network Information

Background: Small molecule-RNA binding sites play a significant role in developing drugs for disease treatment. However, it is a challenge to propose accurate computational tools for identifying these binding sites. Method: In this study, an accurate prediction model named CNRBind was constructed by extracting site significant information from nucleotide and complex networks. We designed complex networks and calculated three topological structural parameters according to RNA tertiary structure. Acknowledging nucleotide interdependence, a sliding window was selected to integrate the influence of adjacent sites. Finally, the model was constructed using a random forest classifier. Results: Compared to the other computational tools, CNRBind was competitive and had excellent discriminative ability for metal ion-binding site prediction. Furthermore, statistic analysis revealed significant differences between CNRBind and existing methods. Additionally, CNRBind is a promising predictor in cases where experimental tertiary structure is unavailable. Conclusion: These results show that CNRBind is effective because of the proposed site significant information encoding strategy. The approach provides a reasonable supplement for biology researches. The dataset and resource codes can be accessed at: https://github.com/Kangxiaoneuq/CNRBind.

Selection Across the Three-Dimensional Structure of Venom Proteins from North American Scolopendromorph Centipedes.

Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution.

Characterization of latently infected EBV+ antibody-secreting B cells isolated from ovarian tumors and malignant ascites

IntroductionIntra-tumoral B cells mediate a plethora of immune effector mechanisms with key roles in anti-tumor immunity and serve as positive prognostic indicators in a variety of solid tumor types, including epithelial ovarian cancer (EOC). Several aspects of intra-tumoral B cells remain unclear, such as their state of activation, antigenic repertoires, and capacity to mature into plasma cells.MethodsB lymphocytes were isolated from primary EOC tissue and malignant ascites and were maintained in cell culture medium. The stably maintained cell lines were profiled with flow cytometry and B cell receptor sequencing. Secreted antibodies were tested with a human proteome array comprising more than 21,000 proteins, followed by ELISA for validation. Originating tumor samples were used for spatial profiling with chip cytometry.ResultsAntibody-secreting B lymphocytes were isolated from the ovarian tumor microenvironment (TME) of four different EOC patients. The highly clonal cell populations underwent spontaneous immortalization in vitro, were stably maintained in an antibody-secreting state, and showed presence of Epstein-Barr viral (EBV) proteins. All originating tumors had high frequency of tumor-infiltrating B cells, present as lymphoid aggregates, or tertiary lymphoid structures. The antigens recognized by three of the four cell lines are coil-coil domain containing protein 155 (CCDC155), growth factor receptor-bound protein 2 (GRB2), and pyruvate dehydrogenase phosphatase2 (PDP2), respectively. Anti-CCDC155 circulating IgG antibodies were detected in 9 of 20 (45%) of EOC patients’ sera. Tissue analyses with multiparameter chip cytometry shows that the antibodies secreted by these novel human B cell lines engage their cognate antigens on tumor cells.DiscussionThese studies demonstrate that within the tumor-infiltrating lymphocyte population in EOC resides a low frequency population of antibody-secreting B cells that have been naturally exposed to EBV. Once stably maintained, these novel cell lines offer unique opportunities for future studies on intratumor B cell biology and new target antigen recognition, and for studies on EBV latency and/or viral reactivation in the TME of non-EBV related solid tumors such as the EOC.

Three-Dimensional Structure of Human Epididymis Protein 4 (HE4): A Protein Modelling of an Ovarian Cancer Biomarker Through In Silico Approach

The Human Epididymis Protein 4 (HE4) biomarker has been extensively investigated for its potential in diagnosing ovarian cancer (OC). For the application of diagnostic techniques and drug delivery, it is crucial to understand the protein tertiary structure. However, the Protein Data Bank (PDB) does not currently contain the three-dimensional (3D) structure of HE4. Therefore, an in silico analysis was conducted to model the HE4 protein using AlphaFold, I-TASSER, and Robetta servers, with the sequence retrieved from UniProt (ID: Q14508). These three servers employed deep learning algorithms, threading templates, and de novo methods, respectively. Subsequently, Molecular Dynamics (MD) simulation using the GROMACS software package improved each 3D structure model, resulting in optimized and refined structures: RF1, RF2, and RF3. PROCHECK and ERRAT programs were employed to assess the structure quality. The Ramachandran plots from PROCHECK indicated that 100% of residues were within the allowed regions for all servers except for I-TASSER. For the refined structures, RF1 and RF3, all residues were concentrated within the allowed regions. According to the ERRAT program, the RF1 model exhibited the highest overall quality factor of 97.701, followed by RF3 and AlphaFold models with scores of 94.643 and 93.750, respectively. After these validations, RF1 emerged as the most accurately predicted 3D structure of HE4 and has one tunnel identified by CAVER 3.0 tool that facilitates the transportation of small particles to the active site, supported by FTsite and PrankWeb binding site predictions. This model holds potential for various computational studies, including the development of OC diagnostic kits. It will enhance our comprehension of the interactions between the protein and other biomolecules.

Unveiling a Comprehensive Multi-epitope Subunit Vaccine Strategy Against Salmonella subsp. enterica: Bridging Core, Subtractive Proteomics, and Immunoinformatics.

Salmonella subsp. enterica (SE) presents a significant global health challenge in both developed and developing countries. Current SE vaccines have limitations, targeting specific strains and demonstrating moderate efficacy in adults, while also being unsuitable for young children and often unaffordable in regions with lower income levels where the disease is prevalent. To address these challenges, this study employed a computational approach integrating core proteomics, subtractive proteomics, and immunoinformatics to develop a universal SE vaccine and identify potential drug targets. Analysis of the core proteome of 185 SE strains revealed 1964 conserved proteins. Subtractive proteomics identified 9 proteins as potential vaccine candidates and 41 as novel drug targets. Using reverse vaccinology-based immunoinformatics, four multi-epitope-based subunit vaccine constructs (MESVCs) were designed, aiming to stimulate cytotoxic T lymphocyte, helper T lymphocyte, and linear B lymphocyte responses. These constructs underwent comprehensive evaluations for antigenicity, immunogenicity, toxicity, hydropathicity, and physicochemical properties. Predictive modeling, refinement, and validation were conducted to determine the secondary and tertiary structures of the SE-MESVCs, followed by docking studies with MHC-I, MHC-II, and TLR4 receptors. Molecular docking assessments showed favorable binding with all three receptors, with SE-MESVC-4 exhibiting the most promising binding energy. Molecular dynamics simulations confirmed the binding affinity and stability of SE-MESVC-4 with the TLR4/MD2 complex. Additionally, codon optimization and in silico cloning verified the efficient translation and successful expression of SE-MESVC-4 in Escherichia coli (E. coli) str. K12. Subsequent in silico immune simulation evaluated the efficacy of SE-MESVC-4 in triggering an effective immune response. These results suggest that SE-MESVC-4 may induce both humoral and cellular immune responses, making it a potential candidate for an effective SE vaccine. However, further experimental investigations are necessary to validate the immunogenicity and efficacy of SE-MESVC-4, bringing us closer to effectively combating SE infections.

Bioinformatics Analysis of 5HT2C Gene Cys23ser Polymorphism and Epigenetics Insights into the HTR2C Receptor Gene Regulation: Implications for Physiological Roles in the Brain

The expression profile, signaling and neuronal functions of the 5-HT2C receptor makes it a candidate of interest for the treatment of several neuropsychiatric diseases. The encoded ligand-gated G-protein coupled receptor (GPCR) protein responds to cell signaling through several neurotransmitters. Signal transduction through G-proteins is a prominent feature of several eukaryotes. In this review and analysis paper, we provide background literature on the unique biochemical, structural, and pharmacological properties of the 5-HT2c receptor and gene architecture, regulation, RNA editing and association studies of polymorphisms.Also on the evolutionary, and phylogenetic paths. of the 5HT2C gene. We conduct in silico predictions using bioinformatics tools SIFT and POLYPHEN to assess the effect of Cys23ser substitution in the N-terminal extracellular loop. Further, epigenetic analysis of the promoter and regions flanking the exon such as (DNase I Hypersensitivity, enhancer with TF binding site); methylation analysis of promoter (CpG methylation and CpG island); and histone marks with FAIRE. The results suggested that the cys23ser substitution can affect the 3D-protein structure. The additional cysteine amino acids (position-23,235,341) in human receptor could enable additional structural stability to the protein and may have evolved from previous ancestors (various mammals and primates) to aid the modulation of behavioral traits under evolutionary pressure. The alterations in DNA methylation and associated regulatory elements in promoter and upstream could impact gene expression, inactivation, genome stabilization, and inheritance which could have relevance in pathological states in the Central nervous system (CNS).

De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure

<i>De Novo</i> Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure

The impact of tertiary lymphoid structures on tumor prognosis and the immune microenvironment in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a common malignancy whose prognosis and treatment outcome are influenced by many factors. Some studies have found that tertiary lymphoid structures (TLSs) in cancer may contribute to prognosis and the prediction of immunotherapy efficacy However, the combined role of TLSs in NSCLC remains unclear. We accessed The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to obtain mRNA sequencing data and clinical information as the TCGA cohort, and used our own sample of 53 advanced NSCLC as a study cohort. The samples were divided into TLS+ and TLS- groups by pathological tissue sections. Patients of the TLS+ group had a better OS (p = 0.022), PFS (p = 0.042), and DSS (p = 0.004) in the TCGA cohort, and the results were confirmed by the study cohort (PFS, p = 0.012). Furthermore, our result showed that the count and size of TLSs are closely associated with the efficacy of immunotherapy. In addition, the TLS+ group was associated with better immune status and lower tumor mutation load. In the tumor microenvironment (TME), the expression levels of CD4+ T cells and CD8+ T cells of different phenotypes were associated with TLSs. Overall, TLSs are a strong predictor of survival and immunotherapeutic efficacy in advanced NSCLC, and T cell-rich TLSs suggest a more ordered and active immune response site, which aids in the decision-making and application of immunotherapy in the clinic.

Design and expression of a chimeric recombinant antigen (SsIR-Ss1a) for the serodiagnosis of human strongyloidiasis: Evaluation of performance, sensitivity, and specificity.

The sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis. The coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest immunogenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively. The preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

Lymphocyte homing and recirculation with tumor tertiary lymphoid structure formation: predictions for successful cancer immunotherapy

The capacity of lymphocytes continuously home to lymphoid structures is remarkable for cancer immunosurveillance and immunotherapy. Lymphocyte homing and recirculation within the tumor microenvironment (TME) are now understood to be adaptive processes that are regulated by specialized cytokines and adhesion molecule signaling cascades. Restricted lymphocyte infiltration and recirculation have emerged as key mechanisms contributing to poor responses in cancer immunotherapies like chimeric antigen receptor (CAR)-T cell therapy and immune checkpoint blockades (ICBs). Uncovering the kinetics of lymphocytes in tumor infiltration and circulation is crucial for improving immunotherapies. In this review, we discuss the current insights into the adhesive and migrative molecules involved in lymphocyte homing and transmigration. The potential mechanisms within the TME that restrain lymphocyte infiltration are also summarized. Advanced on these, we outline the determinates for tertiary lymphoid structures (TLSs) formation within tumors, placing high expectations on the prognostic values of TLSs as therapeutic targets in malignancies.

LARP3, LARP7, and MePCE are involved in the early stage of human telomerase RNA biogenesis

Human telomerase assembly is a highly dynamic process. Using biochemical approaches, we find that LARP3 and LARP7/MePCE are involved in the early stage of human telomerase RNA (hTR) and that their binding to RNA is destabilized when the mature form is produced. LARP3 plays a negative role in preventing the processing of the 3′-extended long (exL) form and the binding of LARP7 and MePCE. Interestingly, the tertiary structure of the exL form prevents LARP3 binding and facilitates hTR biogenesis. Furthermore, low levels of LARP3 promote hTR maturation, increase telomerase activity, and elongate telomeres. LARP7 and MePCE depletion inhibits the conversion of the 3′-extended short (exS) form into mature hTR and the cytoplasmic accumulation of hTR, resulting in telomere shortening. Taken together our data suggest that LARP3 and LARP7/MePCE mediate the processing of hTR precursors and regulate the production of functional telomerase.

Compound heterozygous variants in SLC45A1 might cause syndromic intellectual disability by localization failure and activity attenuation in cells.

Intellectual disability (ID) is a kind of nervous developmental disorder and affects more than 1% of people worldwide. SLC45A1 as a transmembrane protein is implicated in the regulation of glucose homoeostasis. Through trio-based exome sequencing, the missense mutations of SLC45A1 c.103G>A (p.V35M) and c.1211T>G (p.F404C) were identified in the proband with syndromic ID. The distribution, expression and activity of SLC45A1 wild-type (WT) and variants were assayed in transfected COS7 cells. In SLC45A1 variants, the hydrogen bonds surrounding the 35th and 404th amino acid were changed, location on the cytomembrane was failed, their activity to transport glucose was also significantly decreased to contrast with SLC45A1-WT. No difference was observed at the mRNA and protein level. In conclusion, the compound heterozygous variants of SLC45A1 might be the genetic etiology for syndromic ID. These novel mutations probably attenuated its activity to transport glucose by the alteration of tertiary structure and failure of intracellular location.

Accurately Modeling RNA Stem-Loops in an Implicit Solvent Environment.

Ribonucleic acid (RNA) molecules can adopt a variety of secondary and tertiary structures in solution, with stem-loops being one of the more common motifs. Here, we present a systematic analysis of 15 RNA stem-loop sequences simulated with molecular dynamics simulations in an implicit solvent environment. Analysis of RNA cluster ensembles showed that the stem-loop structures can generally adopt the A-form RNA in the stem region. Loop structures are more sensitive, and experimental structures could only be reproduced with modification of CH···O interactions in the force field, combined with an implicit solvent nonpolar correction to better model base stacking interactions. Accurately modeling RNA with current atomistic physics-based models remains challenging, but the RNA systems studied herein may provide a useful benchmark set for testing other RNA modeling methods in the future.

Utilization of an optimized AlphaFold protein model for structure-based design of a selective HDAC11 inhibitor with anti-neuroblastoma activity.

AlphaFold is an artificial intelligence approach for predicting the three-dimensional (3D) structures of proteins with atomic accuracy. One challenge that limits the use of AlphaFold models for drug discovery is the correct prediction of folding in the absence of ligands and cofactors, which compromises their direct use. We have previously described the optimization and use of the histone deacetylase 11 (HDAC11) AlphaFold model for the docking of selective inhibitors such as FT895 and SIS17. Based on the predicted binding mode of FT895 in the optimized HDAC11 AlphaFold model, a new scaffold for HDAC11 inhibitors was designed, and the resulting compounds were tested in vitro against various HDAC isoforms. Compound 5a proved to be the most active compound with an IC50 of 365 nM and was able to selectively inhibit HDAC11. Furthermore, docking of 5a showed a binding mode comparable to FT895 but could not adopt any reasonable poses in other HDAC isoforms. We further supported the docking results with molecular dynamics simulations that confirmed the predicted binding mode. 5a also showed promising activity with an EC50 of 3.6 µM on neuroblastoma cells.

Weak-cooperative binding of a long single-stranded DNA chain on a surface.

Binding gene-wide single-stranded nucleic acids to surface-immobilized complementary probes is an important but challenging process for biophysical studies and diagnostic applications. The challenge comes from the conformational dynamics of the long chain that affects its accessibility and weakens its hybridization to the probes. We investigated the binding of bacteriophage genome M13mp18 on several different 20-mer probes immobilized on the surface of a multi-spot, label-free biosensor, and observed that only a few of them display strong binding capability with dissociation constant as low as 10 pM. Comparing experimental data and computational analysis of the M13mp18 chain structural features, we found that the capturing performance of a specific probe is directly related to the multiplicity of binding sites on the genomic strand, and poorly connected with the predicted secondary and tertiary structure. We show that a model of weak cooperativity of transient bonds is compatible with the measured binding kinetics and accounts for the enhancement of probe capturing observed when more than 20 partial pairings with binding free energy lower than -10 kcal mol-1 are present. This mechanism provides a specific pattern of response of a genomic strand on a panel of properly selected oligomer probe sequences.