Intermediate Binding Affinity Research Articles

Molecular identification and functional analysis of Niemann-Pick type C2 proteins，carriers for semiochemicals and other hydrophobic compounds in the brown dog tick, Rhipicephalus linnaei

Ticks are important vectors of many pathogens with tremendous impact on human and animal health. Studies of semiochemical interactions and mechanisms underlying chemoreception can provide important tools in tick management. Niemann-Pick type C2 (NPC2) proteins have been proposed as one type of chemoreceptor in arthropods. Here, we cloned two NPC2 genes in the brown dog tick, Rhipicephalus linnaei, the tropical lineage previously named R. sanguineus sensu lato and characterized them functionally.R.linNPC2a and R.linNPC2b genes were found to be expressed at each developmental stage with the highest level in adult males. By using quantitative real-time PCR we revealed expression in multiple tissues, including midgut, ovary, salivary glands and legs. Ligand binding analysis revealed that R.linNPC2b bound a wide spectrum of compounds, with β-ionone, α-amylcinnamaldehyde, 2-nitrophenol and benzaldehyde displaying the strongest binding affinity (Ki < 10 μM), whereas R.linNPC2a showed a more narrow ligand binding range, with intermediate binding affinity to α-amylcinnamaldehyde and 2-nitrophenol (Ki < 20 μM). Molecular docking indicated that the amino acid residue Phe89, Leu77 and Val131 of R.linNPC2a and Phe70, Leu132 and Phe73 of R.linNPC2b could bind multiple ligands. These residues might thus play a key role in the identification of the volatiles. Our results contribute to the understanding of olfactory mechanisms of R. linnaei and can offer new pathways towards new management strategies.

Binding Affinity of Monoalkyl Phosphinic Acid Ligands toward Nanocrystal Surfaces

We recently introduced monoalkyl phosphinic acids as a ligand class for nanocrystal (NC) synthesis. Their metal salts have interesting reactivity differences compared to metal carboxylates and phosphonates and provide a cleaner work-up compared to phosphonates. However, little is known about the surface chemistry of NCs with monoalkyl phosphinate ligands. Here, we probe the relative binding affinity of monoalkyl phosphinate ligands with respect to other X-type ligands. We perform competitive ligand exchange reactions with carboxylate and phosphonate ligands at the surface of hafnia, cadmium selenide, and zinc sulfide NCs. We monitor the ligand shell composition by solution 1H and 31P NMR spectroscopy. Using a monoalkyl phosphinic acid with an ether functionality, we gain an additional NMR signature, apart from the typical alkene resonance in oleic acid and oleylphosphonic acid. We find that carboxylate ligands are easily exchanged upon exposure to monoalkyl phosphinic acids, whereas an equilibrium is reached between monoalkyl phosphinates and phosphonates, slightly in the favor of phosphonate (K = 2). Phosphinic acids have thus an intermediate binding affinity between carboxylic acids and phosphonic acids for all the NCs studied. These results enable the sophisticated use of monoalkyl phosphinic acids for NC synthesis and for post-synthetic surface engineering.

Abstract 6130: Comprehensive genomic and transcriptomic profiling of acral lentiginous melanoma

Abstract Background: Acral lentiginous melanoma (ALM) is a rare melanoma subtype found on the palms, soles and nailbeds. Outcomes are poor for patients with advanced ALM, and novel treatment approaches are needed. Here, we seek to explore the global genomic and transcriptomic landscape of ALM. Methods: A total of 699 primary CM (non-ALM cutaneous melanoma) and 18 primary ALM samples underwent next generation sequencing of DNA (592 Gene Panel, NextSeq, or WES, NovaSeq), and whole transcriptome sequencing (NovaSeq, WTS). Wilcoxon, Fisher’s exact test were used to determine statistical significance (displayed as p value without and q value with multi comparison correction). xCell, HLA subtyping, neoantigen load (HBA: high binding affinity; IBA: intermediate binding affinity; LBA: low binding affinity), Interferon gamma score (IFNγ), MAPK pathway activity score (MPAS), and Innate anti-PD-1 Resistance score (IPRES) were calculated by mRNA expression. Global differentially regulated genes were assessed via limma R package (C: log fold change). Results: The most common alterations in ALM included NRAS (22.2%), NF1 (20.0%), BRAF (11.1%) and CDKN2A (11.1%) mutations, and EMSY (22.2%), ELL (11.1%), MAML2 (11.1%), MRE11(11.1%) and PIK3R2 (11.1%) amplifications. ALM had lower TMB (1.5 v 9 Mut/Mb, q&amp;lt;.0001), lower rates of TERT (0 v 66.7%, q&amp;lt;.01) and a trend towards lower BRAF (11.1 v 39.8%, p&amp;lt;.05) mutations, compared to CM. Neoantigen load was lower in ALM compared to CM, regardless of MHC binding affinity (HBA: 1 vs 4, q &amp;lt;.01; IBA: 2 vs 7, q &amp;lt;.001; LBA: 7 vs 18, q &amp;lt;.001). HLA-G RNA expression was upregulated in ALM with respect to CM (C = 1.14, q &amp;lt;.001). ALM showed less CD4+ T cell Th1 (C = -0.8, p &amp;lt;.05), B cell plasma (C = -1.8, p&amp;lt;.05), and γδ T cells (C = -5.9, p&amp;lt;.05), but more CD4+ T cell central memory cell (C = 9.7, p&amp;lt;.05), stroma score (C = 1.7, p&amp;lt;.05), and endothelial cells (C = 1.7, p&amp;lt;.05), versus CM. There was a trend towards lower IFNγ in ALM (-0.4 vs -0.3, p = .1), but no difference in IPRES, compared to CM (-0.09 vs 0.1, p=.9). MPAS scores were lower for ALM compared to CM (-1.6 vs -0.4, q&amp;lt;.001), even when stratifying by BRAF (q&amp;lt;.05) or NF1 (q&amp;lt;.05) status, but not NRAS (p = .22). Pathways related to keratinization (p &amp;lt;.0001) and amyloid fiber formation (p&amp;lt;.0001) were enriched in ALM, due to overexpression of KRT16 (C = 3.5, q &amp;lt;.01), KRT6B (C = 3.4, q &amp;lt;.01), and KRT17 (C = 3.2, q &amp;lt;.05), among others. Conclusion: ALM has distinct immunologic features, including upregulation of HLA-G, as well as lower MAPK activation in ALM, compared to CM, highlighting the need for novel therapeutic approaches in the treatment of this rare subtype. Citation Format: Gino K. In, Jun Yin, Phillip Walker, Justin Moser, Joanne Xiu, Kelsey Poorman, Geoffery T. Gibney, Matthew Oberley, Thuy Phung, Leonel F. Hernandez-Aya, Jose Lutzky, Wolfgang Michael Korn, Michael B. Atkins. Comprehensive genomic and transcriptomic profiling of acral lentiginous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6130.

A Novel Humanized Murine Model to Identify Neoantigen-Specific T Cells in CBFA2T3-GLIS2 Positive Acute Megakaryoblastic Leukemia

CBFA2T3-GLIS2 is an oncogenic fusion gene that acts as the driver mutation in ~18% of pediatric patients with acute megakaryoblastic leukemia (AMKL) that do not have Down syndrome. CBFA2T3-GLIS2 driven leukemia carries the worst prognosis among all pediatric acute myeloid (AML) subtypes, with event free and overall survival probabilities of 8% and 14% respectively. Chemotherapy and stem cell transplantation fail to cure this disease, thus innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven't been realized in AML due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which use heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by MHC molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, we established a humanized patient derived xenograft (PDX) murine model by conditioning 15 immunodeficient NSG-SGM3 mice with 200 rads 24 hours prior to transplantation of 3.5 million CBFA2T3-GLIS2 positive PDX cells derived from an individual female patient. After 20 days of engraftment, 2.5 million HLA class I matched PBMCs from an unrelated female donor were introduced. Experimental mice (engrafted with both PDX cells and PBMCs), leukemia only control mice, and PBMC only control mice were sacrificed at days 30, 35, and 40. Flow cytometry analysis at day 40 showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to the leukemia only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at day 40 in the PDX mice with PBMCs compared to the PBMC only control (9665.4 pg/ml vs. 789.7 pg/ml, p<0.001), indicating a potential leukemia-directed cytokine lymphocyte response. Single cell TCR repertoire analysis of CD8+ T cells from day 40 revealed a decrease in alpha beta TCR diversity when exposed to PDX cells compared to the PBMC only control, consistent with a clonal expansion of neoantigen-directed TCRs. Single TCR expressing stable cell lines have been generated for the top expanded clones. In ongoing experiments, we are utilizing these cell lines to assess leukemia clearance in our model and measuring specificity and functionality for the putative neoantigens through tetramer staining and peptide stimulation assays, respectively. For the latter studies, we have taken two approaches for the identification of potential neoantigens that may be directing this observed clonal expansion. First, to interrogate potential neoantigens spanning the fusion junction we used the algorithm from NetMHCcons which identified two peptides with intermediate binding affinity (defined by an IC50 of 150 - 500 nM). Second, we utilized immunopeptidomics to identify HLA class I presented peptides on the surface of the PDX cells. One of our top significant hits was a peptide that corresponds to the 5‘ untranslated region (UTR) of bone morphogenetic protein 2 (BMP2). This peptide fragment was not present in the human Uniprot database, suggesting the 5‘ UTR is not expressed in normal tissues. Although its role in leukemia is not well described, BMP2 is aberrantly upregulated in CBFA2T3-GLIS2 positive AMKL and required for serial replating in colony formation unit assays; therefore, this peptide is of great interest as a candidate leukemia-associated antigen. In summary, our novel humanized PDX model sheds light on the immunogenicity of CBFA2T3-GLIS2 mediated AMKL and the potential for TCR-T immunotherapy in the setting of minimal residual disease for this chemotherapy resistant malignancy. Studies are ongoing to identify the TCR clone(s) responsible for the anti-leukemic response seen in our model and the leukemia-associated antigens that are mediating this response. DisclosuresGruber: Kura Oncology: Consultancy.

Enhanced Drug Delivery to Solid Tumors via Drug-Loaded Nanocarriers: An Image-Based Computational Framework.

ObjectiveNano-sized drug delivery systems (NSDDSs) offer a promising therapeutic technology with sufficient biocompatibility, stability, and drug-loading rates towards efficient drug delivery to solid tumors. We aim to apply a multi-scale computational model for evaluating drug delivery to predict treatment efficacy.MethodologyThree strategies for drug delivery, namely conventional chemotherapy (one-stage), as well as chemotherapy through two- and three-stage NSDDSs, were simulated and compared. A geometric model of the tumor and the capillary network was obtained by processing a real image. Subsequently, equations related to intravascular and interstitial flows as well as drug transport in tissue were solved by considering real conditions as well as details such as drug binding to cells and cellular uptake. Finally, the role of periodic treatments was investigated considering tumor recurrence between treatments. The impact of different parameters, nanoparticle (NP) size, binding affinity of drug, and the kinetics of release rate, were additionally investigated to determine their therapeutic efficacy.ResultsUsing NPs considerably increases the fraction of killed cells (FKCs) inside the tumor compared to conventional chemotherapy. Tumoral FKCs for two-stage DDS with smaller NP size (20nm) is higher than that of larger NPs (100nm), in all investigate release rates. Slower and continuous release of the chemotherapeutic agents from NPs have better treatment outcomes in comparison with faster release rate. In three-stage DDS, for intermediate and higher binding affinities, it is desirable for the secondary particle to be released at a faster rate, and the drug with slower rate. In lower binding affinities, high release rates have better performance. Results also demonstrate that after 5 treatments with three-stage DDS, 99.6% of tumor cells (TCs) are killed, while two-stage DDS and conventional chemotherapy kill 95.6% and 88.5% of tumor cells in the same period, respectively.ConclusionThe presented framework has the potential to enable decision making for new drugs via computational modeling of treatment responses and has the potential to aid oncologists with personalized treatment plans towards more optimal treatment outcomes.

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C44), 83% carbamylated (C83) and 85% acetylated (A85) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C83 and A85 HSAs while C44 HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (KSV) and bimolecular quenching rate constant (kq) suggested, NA-HSA complex formation, binding constant (Ka) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the Ka value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.

Characterization of the interaction of diosgenin with human serum albumin and α1-acid glycoprotein using biophysical and bioinformatic tools

Diosgenin (DIO), a steroidal sapogenin with high therapeutic potential, has been shown to exhibit a myriad of pharmacologically significant properties and is commonly used as a template for synthesizing steroidal drugs. The efficacy of bioactive compounds as a potential drug depends mainly upon their interaction with proteins in the circulatory system. Therefore, the interaction of DIO with the major carrier proteins in humans, i.e. human serum albumin (HSA) and α 1 -acid glycoprotein (AAG), was characterized using multispectroscopic and molecular docking techniques. Analysis of fluorescence enhancement data showed intermediate binding affinity for both DIO–HSA and DIO–AAG interactions. Circular dichroism spectral results revealed minimal conformational alterations in HSA upon DIO binding, but the effect on AAG was more significant. Competitive ligand displacement results suggested that DIO does not bind strongly to the main drug binding sites of HSA. However, DIO was found to favor binding to subdomain IB of HSA based on molecular docking data. For AAG, the binding location of DIO was determined for the two variants of AAG (A and F1*S variants) using molecular docking. For variant A, the binding site was situated in lobes I and II of the central binding pocket of the protein, while in variant F1*S, DIO was docked to a cleft on the protein surface. Hydrophobic interactions and van der Waals forces were mainly responsible for complexation between DIO and both proteins. Additionally, there was involvement of hydrogen bonding between DIO and both variants of AAG, but not between DIO and HSA. • AAG binds to DIO with slightly stronger affinity than HSA. • Hydrophobic forces are mainly responsible for DIO binding to both proteins. • DIO binds to HSA at subdomain IB. • DIO binds to variant F1*S of AAG at a cleft at the surface. • DIO binds to variant A of AAG at the central cavity.

Eukaryotic transcription factors can track and control their target genes using DNA antennas

Eukaryotic transcription factors (TF) function by binding to short 6-10 bp DNA recognition sites located near their target genes, which are scattered through vast genomes. Such process surmounts enormous specificity, efficiency and celerity challenges using a molecular mechanism that remains poorly understood. Combining biophysical experiments, theory and bioinformatics, we dissect the interplay between the DNA-binding domain of Engrailed, a Drosophila TF, and the regulatory regions of its target genes. We find that Engrailed binding affinity is strongly amplified by the DNA regions flanking the recognition site, which contain long tracts of degenerate recognition-site repeats. Such DNA organization operates as an antenna that attracts TF molecules in a promiscuous exchange among myriads of intermediate affinity binding sites. The antenna ensures a local TF supply, enables gene tracking and fine control of the target site’s basal occupancy. This mechanism illuminates puzzling gene expression data and suggests novel engineering strategies to control gene expression.

Biophysical and computational approaches to unravel the molecular interaction mechanism of bromodeoxyuridine, a proliferative marker with human serum albumin

The interaction of a nucleoside analogue bromodeoxyuridine (BrdU) with human serum albumin (HSA) was studied to investigate the binding phenomenon and analyse the protein conformation upon BrdU binding. Multiple spectroscopic techniques, viz. intrinsic and 3-D fluorescence, UV–Vis absorption, and circular dichroism (CD) spectroscopy along with molecular docking were used. Decrease in the Stern–Volmer constant (Ksv) as well as the association constant (Ka) with increase in temperature suggested BrdU–HSA complex formation. Intermediate binding affinity between BrdU and HSA was evident from the Ka values (2.49–3.97 × 104 mol–1 dm3), while BrdU–HSA complex formation was driven by hydrophobic and van der Waals interactions along with hydrogen bonds, as revealed by thermodynamic data (ΔS = + 28.48 J mol−1 K−1; ΔH = − 17.16 kJ mol−1). Minor changes occur in both secondary and tertiary structures as well as in the fluorophores’ microenvironment of HSA, as recognized from the CD spectral results in the far-UV and the near-UV regions and 3-D fluorescence spectra, respectively. Use of site markers (warfarin and indomethacin for site I; diazepam for site II) as well as docking results suggested BrdU binding to both site I (more preferred) and site II, located in subdomains IIA and IIIA, respectively, of HSA.

Exploring the interaction between tyrphostin 9 and human serum albumin using biophysical and computational methods

Tyrphostin 9 (Tyr 9) is a potent platelet-derived growth factor receptor (PDGFR) inhibitor, which induces apoptosis in various cancer cell types. The binding of Tyr 9 to the major transport protein, human serum albumin (HSA) was investigated using several spectroscopic techniques and molecular docking method. Fluorescence quenching titration results showed progressive decrease in the protein fluorescence with increasing drug concentrations. A decreasing trend of the Stern-Volmer constant, K sv with increasing temperature characterized the drug-induced quenching as static quenching, thus pointed towards the formation of Tyr 9–HSA complex. The binding constant of Tyr 9–HSA interaction was found to lie within the range 3.48–1.69 × 105 M−1 at three different temperatures, i.e. 15 °C, 25 °C and 35 °C, respectively and suggested intermediate binding affinity between Tyr 9 and HSA. The drug–HSA complex seems to be stabilized by hydrophobic forces, van der Waals forces and hydrogen bonds, as suggested from the thermodynamic data as well as molecular docking results. The far-UV and the near-UV CD spectral results showed slight alteration in the secondary and tertiary structures, respectively, of the protein upon Tyr 9 binding. Interaction of Tyr 9 with HSA also produced microenvironmental perturbations around protein fluorophores, as evident from the three-dimensional fluorescence spectral results but increased protein’s thermal stability. Both competitive drug binding results and molecular docking analysis suggested Sudlow’s Site I of HSA as the preferred Tyr 9 binding site. Communicated by Ramaswamy H. Sarma

T cell recognition of novel shared breast cancer antigens is frequently observed in peripheral blood of breast cancer patients

ABSTRACTAdvances within cancer immunotherapy have fueled a paradigm shift in cancer treatment, resulting in increasing numbers of cancer types benefitting from novel treatment options. Despite originally being considered an immunologically silent malignancy, recent studies encourage the research of breast cancer immunogenicity to evaluate immunotherapy as a treatment strategy. However, the epitope landscape in breast cancer is minimally described, limiting the options for antigen-specific, targeted strategies. Aromatase, never in mitosis A-related kinase 3 (NEK3), protein inhibitor of activated STAT3 (PIAS3), and prolactin are known as upregulated proteins in breast cancer. In the present study, these four proteins are identified as novel T cell targets in breast cancer.From the four proteins, 147 peptides were determined to bind HLA-A*0201 and -B*0702 using a combined in silico/in vitro affinity screening. T cell recognition of all 147 peptide-HLA-A*0201/-B*0702 combinations was assessed through the use of a novel high-throughput method utilizing DNA barcode labeled multimers.T cell recognition of sequences within all four proteins was demonstrated in peripheral blood of patients, and significantly more T cell responses were detected in patients compared to healthy donors for both HLA-A*0201 and -B*0702. Notably, several of the identified responses were directed toward peptides, with a predicted low or intermediate binding affinity. This demonstrates the importance of including low-affinity binders in the search for epitopes within shared tumor associated antigens (TAAs), as these might be less subject to immune tolerance mechanisms.The study presents four novel TAAs containing multiple possible targets for immunotherapy of breast cancer.

Strength of T cell signaling regulates HIV-1 replication and establishment of latency.

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir.

Molecular interaction study of an anticancer drug, ponatinib with human serum albumin using spectroscopic and molecular docking methods.

Binding of a potent anticancer agent, ponatinib (PTB) to human serum albumin (HSA), main ligand transporter in blood plasma was analyzed with several spectral techniques such as fluorescence, absorption and circular dichroism along with molecular docking studies. Decrease in the KSV value with increasing temperature pointed towards PTB-induced quenching as the static quenching, thus affirming complexation between PTB and HSA. An intermediate binding affinity was found to stabilize the PTB-HSA complex, as suggested by the Ka value. Thermodynamic analysis of the binding phenomenon revealed participation of hydrophobic and van der Waals interactions along with hydrogen bonds, which was also supported by molecular docking analysis. Changes in both secondary and tertiary structures as well as in the microenvironment around Trp and Tyr residues of HSA were anticipated upon PTB binding to the protein, as manifested from circular dichroism and three-dimensional fluorescence spectra, respectively. Binding of PTB to HSA led to protein's thermal stabilization. Competitive ligand displacement experiments using different site markers such as warfarin, indomethacin and ketoprofen disclosed the binding site of PTB as Sudlow's site I in HSA, which was further confirmed by molecular docking analysis.

Biophysical characterization and ligand-binding properties of the elongation factor Tu from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is the key devastating bacterial pathogen responsible for tuberculosis. Increasing emergence of multi-drug-resistant, extensively drug-resistant, and rifampicin/isoniazid-resistant strains of Mtb makes the discovery of validated drug targets an urgent priority. As a vital translational component of the protein biosynthesis system, elongation factor Tu (EF-Tu) is an important molecular switch responsible for selection and binding of the cognate aminoacyl-tRNA to the acceptor site on the ribosome. In addition, EF-Tu from Mtb (MtbEF-Tu) is involved in the initial step of trans-translation which is an effective system for rescuing the stalled ribosomes from non-stop translation complexes under stress conditions. Given its crucial role in protein biosynthesis, EF-Tu is identified as an excellent molecular target for drug design. Here, we reported the recombinant expression, purification, biophysical characterization, and structural modeling of the MtbEF-Tu protein. Our results demonstrated that prokaryotic expression plasmids of pET28a-MtbEF-Tu could be expressed efficiently in Escherichia coli. We successfully purified the 6× His-tagged proteins with a yield of 16.8 mg from 1 l of Luria Bertani medium. Dynamic light scattering experiments showed that MtbEF-Tu existed in a monomeric form, and circular dichroism experiments indicated that MtbEF-Tu was well structured. Moreover, isothermal titration calorimetry experiments displayed that the purified MtbEF-Tu protein possessed intermediate binding affinities for guanosine-5'-triphosphate (GTP) and GDP. The GTP/GDP-binding sites were predicted by flexible molecular docking approach which reveals that GTP/GDP binds to MtbEF-Tu mainly through hydrogen bonds. Our work lays the essential basis for further structural and functional studies of MtbEF-Tu as well as MtbEF-Tu-related novel drug developments.

Abstract B33: Neoepitope peptide vaccines and immune checkpoint blockade in a new preclinical ovarian cancer model

Abstract Objective: Therapeutic success currently seen with immune checkpoint blockade (ICB) occurs primarily in immunogenic tumors and can be partly explained by the high rates of non-synonymous genetic mutations (single nucleotide variants, SNV) in tumor cells, leading to neoepitope presentations and robust T-cell infiltration. Despite showing mutations in fewer genes (most frequently TP53), high-grade serous ovarian cancer (HGSOC) displays signs of T-cell inflammation and responds, albeit modestly, to ICB. We focus on immune therapy tailored to the genomic characteristics of the tumor, especially to structural aberrations (SAs), like gene fusions. Our hypothesis is that SAs that are expressed as proteins are antigenic and immunogenic, and represent a source of neoantigens for combination immunotherapies that can overcome locoregional immune suppression. DNA structural aberrations can result in intergenic fusions and intragenic rearrangements, such as exon duplications (dup) and deletions (del). Here we focus on identifying gene fusion and exon dup/del-derived neoepitopes based on RNA-seq data for a combination of retrospective and prospective sample collections. Our hypothesis is that these gene rearrangements resulting from SVs may contribute to the immunogenicity and antigenicity of HGSOC. Methods: FusionCatcher was used to evaluate RNA sequencing data for in-frame gene fusions events in banked human HGSOC. HLAminer was used to determine individual haplotypes. Upon detection of fusion events (validated via PCR) we identified the MHC-I and MHC-II restricted peptides with predicted high or intermediate (IC50&amp;lt;500nM) binding affinity. The peptides (with sequences across the fusion point) were synthesized and pulsed onto HLA-matched PBMCs, for in vitro T cells stimulation assay. Following antigen priming and in vitro restimulation, IFNγ production by T cells was measured by ELISA. The efficacy of SA-derived neoepitopes was tested in vivo with two new preclinical mouse models based on MKP-Lung and MKP-Liver cells ovarian cancer cell lines. RNA-seq data of their complete mutanome were used to generate neoepitopes. RNA from normal mouse OSE isolated from healthy mice was used as reference for mutation calling. For vaccine generation, peptides were pulsed onto dendritic cells matured into Th1-inducing phenotype (DC1). Mice were treated with Rat IgG, anti-PDL1, or vaccine + anti PD-L1. Objective response was measured by tumor volume. Results: Our results demonstrate neoepitopes-specific human T-cell responses in vitro. RNA-seq data were available for nine patients; eight MHC-I and five MHC-II neoantigens were synthesized from gene fusions from three different patients represented by six sets of haplotype specific PBMCs. In vitro, neoantigen-treated PBMCs showed increased cell clumping. An objective IFN gamma response on ELISA was seen for six peptides at two different concentrations and five peptides at one concentration. Analysis of RNA-seq of murine tumors shows that the metastatic MKP-Lung cells (which carry molecular signatures associated with recurrent ovarian cancer), have an increased number of mutations (SNVs, indels) including gene fusions, in line with findings from human tumors. Using in silico prediction tools, we identified 16 potential peptide binders (IC50&amp;lt; 500), two with strong and 14 with moderate binding. The transcript variants were validated via PCR. The vaccine comprised the top seven MHC-I restricted peptide candidates with IC50&amp;lt;500Nm and one large (MHC-II) candidate peptide for CD4 T-cell responses. No difference was seen between treatment groups (ANOVA p=0.35). Conclusion: Our results demonstrate that PBMC pulsed with a combination of short and large peptides release increased IFNγ, supporting the proposed vaccine design. Structural variants contribute to tumor immunogenic potential. Immunogenicity data from this project will be further used to refine the target selection for individualized, mutanome-based vaccines. Citation Format: Malcolm S. Ross, Ma Tianzhou, Lixin Zhang, Nolan Priedigkeit, George Tseng, Adrian V. Lee, Robert P. Edwards, Anda M. Vlad. Neoepitope peptide vaccines and immune checkpoint blockade in a new preclinical ovarian cancer model. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B33.

Intermolecular interaction of fosinopril with bovine serum albumin (BSA): The multi-spectroscopic and computational investigation.

The intermolecular interaction of fosinopril, an angiotensin converting enzyme inhibitor with bovine serum albumin (BSA), has been investigated in physiological buffer (pH7.4) by multi-spectroscopic methods and molecular docking technique. The results obtained from fluorescence and UV absorption spectroscopy revealed that the fluorescence quenching mechanism of BSA induced by fosinopril was mediated by the combined dynamic and static quenching, and the static quenching was dominant in this system. The binding constant, Kb , value was found to lie between 2.69×103 and 9.55×103 M-1 at experimental temperatures (293, 298, 303, and 308K), implying the low or intermediate binding affinity between fosinopril and BSA. Competitive binding experiments with site markers (phenylbutazone and diazepam) suggested that fosinopril preferentially bound to the site I in sub-domain IIA on BSA, as evidenced by molecular docking analysis. The negative sign for enthalpy change (ΔH0 ) and entropy change (ΔS0 ) indicated that van der Waals force and hydrogen bonds played important roles in the fosinopril-BSA interaction, and 8-anilino-1-naphthalenesulfonate binding assay experiments offered evidence of the involvements of hydrophobic interactions. Moreover, spectroscopic results (synchronous fluorescence, 3-dimensional fluorescence, and Fourier transform infrared spectroscopy) indicated a slight conformational change in BSA upon fosinopril interaction.

Elucidating the 16S rRNA 3\u2032 boundaries and defining optimal SD/aSD pairing in Escherichia coli and Bacillus subtilis using RNA-Seq data

Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5′ UTR of mRNA and the anti-SD (aSD) sequence at the free 3′ end of the 16S rRNA (3′ TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3′ TAIL and 2) extent of the core aSD sequence within the 3′ TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves 1) mapping the 3′ boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3′ TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3′ TAIL in B. subtilis and recovered the established 3′ TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein.

Mining the Proteome of Leishmania donovani for the Development of Novel MHC Class I Restricted Epitope for the Control of Visceral Leishmaniasis.

Although, the precise host defence mechanism(s) is not completely understood, T cell-mediated immune responses is believed to play a pivotal role in controlling parasite infection. Here we target the stage dependent over expressed gene. Here, the consensus based computational approach was adopted for the screening of potential major histocompatibility complex class I restricted epitopes. Based on the computational analysis and previously published report, a set 19 antigenic proteins derived from Leishmania donovani were screened for further characterization as vaccine candidates. A total of 49 epitopes were predicted, which revealed a comprehensive binding affinity to the 40 different MHC class I supertypes. Based on the population coverage and HLA cross presentation, nine highly promiscuous epitopes such as LTYDDVWTV (P1), FLFPQRTAL(P2), FLFSNGAVV (P3), YIYNFGIRV (P4), YMTAAFAAL (P5), KLLRPFAPL (P6), FMLGWIVTI (P7), SLFERNKRV (P8), and SVWNRIFTL (P9) which have either a high or an intermediate TAP binding affinity were selected for further analysis. Theoretical population coverage analysis of polytope vaccine (P1-P9) revealed more than 92% population. Stimulation with the cocktail of peptide revealed a proliferative CD8+ T cell response and increased IFN-γ production. An upregulated NF-κB activity is thought to be play a pivotal role in T cell proliferation against the selected peptide. The Th1-type cytokine profile (presence of IFN-γ and absence of IL-10) suggests the potentiality of the cocktail of epitope as a subunit vaccine against leishmaniasis. However, the efficiency of these epitopes to trigger other Th1 cytokines and chemokines in a humanized mice model could explore its plausibility as a vaccine candidate. J. Cell. Biochem. 119: 378-391, 2018. © 2017 Wiley Periodicals, Inc.

Structural Basis of Egg Coat-Sperm Recognition at Fertilization

SummaryRecognition between sperm and the egg surface marks the beginning of life in all sexually reproducing organisms. This fundamental biological event depends on the species-specific interaction between rapidly evolving counterpart molecules on the gametes. We report biochemical, crystallographic, and mutational studies of domain repeats 1–3 of invertebrate egg coat protein VERL and their interaction with cognate sperm protein lysin. VERL repeats fold like the functionally essential N-terminal repeat of mammalian sperm receptor ZP2, whose structure is also described here. Whereas sequence-divergent repeat 1 does not bind lysin, repeat 3 binds it non-species specifically via a high-affinity, largely hydrophobic interface. Due to its intermediate binding affinity, repeat 2 selectively interacts with lysin from the same species. Exposure of a highly positively charged surface of VERL-bound lysin suggests that complex formation both disrupts the organization of egg coat filaments and triggers their electrostatic repulsion, thereby opening a hole for sperm penetration and fusion.

Antidepressants and risk of cataract development: A population-based, nested case-control study

BackgroundPrevious studies demonstrated increased risk of cataract development among users of selective serotonin reuptake inhibitors (SSRIs). However, it remains unknown whether this risk also prevails with the use of other types of antidepressants. The aim of this study was to investigate whether use of antidepressants is associated with an increased risk of cataract development. Moreover, the relationship between binding affinities of serotonin transporter (SERT) of antidepressant and the risk of cataracts is examined. MethodsWe conducted a nested case-control study using National Health Insurance Research Database in Taiwan. A total of 14,288 patients were included; 7651 in the cataract group and 6637 in the control group. Antidepressant exposure was categorized by type, duration of use, and binding affinities of SERT. The association between antidepressant exposure and cataract development was assessed using conditional logistic regression analysis. ResultsThe adjusted odds ratios (AORs) for developing cataracts among continuous users of SSRIs, serotonin norepinephrine reuptake inhibitors (SNRIs), and other antidepressants were 1.26 (95% confidence interval (CI): 1.12–1.41, p<0.001), 1.21 (95% CI: 1.02–1.43, p=0.027), and 1.18 (95% CI: 1.04–1.34, p=0.009) respectively. Specifically, continuous uses of fluoxetine (AOR: 1.21; 95% CI: 1.01–1.46, p=0.042), fluvoxamine (AOR: 1.47; 95% CI: 1.01–2.12, p=0.043), venlafaxine (AOR: 1.44; 95% CI: 1.19–1.74, p<0.001) significantly increased the risk of cataract development. Moreover, continuous users of antidepressants with intermediate SERT binding affinities (AOR: 1.68; 95% CI: 1.10–2.56, p=0.017) were significantly associated with increased risks of cataract development. LimitationsSeveral confounding factors such as obesity, multiple drug users, family history of cataracts, substance use, and environmental factors (such as sunlight or radiation exposure) were acquired. ConclusionsWe found increased risk of cataract development in patients continuously using antidepressants. Regular ocular evaluations in these patients are warranted.