Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea β-Vignin during Seed Germination

The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein β-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of β-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of β-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. β-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.

The iterative application of a large chemical space in the drug discovery process

Aim. To demonstrate the advantages of large-scale virtual libraries generated using chemical protocols previously validated in primary steps of the drug discovery process.Results and discussion. Two validated parallel chemistry protocols reported earlier were used to create the chemical space. It was then sampled based on diversity metric, and the sample was subjected to the virtual screening on BRD4 target. Hits of virtual screening were synthesized and tested in the thermal shift assay.Experimental part. The chemical space was generated using commercially available building blocks and synthetic protocols suitable for parallel chemistry and previously reported. After narrowing it down, using MedChem filters, the resulting sub-space was clustered based on diversity metrics. Centroids of the clusters were put to the virtual screening against the BRD4 active center. 29 Hits from the docking were synthesized and subjected to the thermal shift assay with BRD4, and 2 compounds showed noticeable dTm.Conclusions. A combination of cheminformatics and molecular docking was applied to find novel potential binders for BRD4 from a large chemical space. The selected set of predicted molecules was synthesized with a 72 % success rate and tested in a thermal shift assay to reveal a 6 % hit rate. The selection can be performed iteratively to fast support of the drug discovery.

Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling

Despite the growing interest in using chemical genetics in plant research, small-molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass-spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or post-translational modifications allows the identification of drug targets, or the study of protein-metabolite and protein-protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal- and the phospho-proteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf thus, revealing a novel crosstalk between brassinosteroid and auxin signaling.

Targeting the Oncogenic Functions of an RNA-Binding Protein Involved in Hematologic Malignancies

hnRNP K (heterogeneous ribonucleoprotein K) is an RNA-binding protein that binds to conserved poly-C rich tracks in RNA and influences a diverse set of molecular pathways involved in tumorigenesis. Our previous studies identified hnRNP K overexpression in patients with diffuse large B-cell lymphoma (46/75, 61%) and acute myeloid leukemia (45/160, 28%). This overexpression correlates with dismal clinical outcomes and a lack of therapeutic responses to standard treatment. To explore hnRNP K's in vivo functions, we generated Hnrnpk-transgenic mouse models. These mice develop lymphoma phenotypes through activation of the c-Myc pathway. In pre-clinical settings, bromodomain inhibitors disrupted hnRNP K-mediated c-Myc activation, demonstrating that hnRNP K overexpression mediated-pathways are amenable to therapeutic intervention. To further our studies, we used IP-mass spectrometry, RNA-sequencing, RNA immunoprecipitation, reverse phase protein analyses, and polysome profiling to identify novel pathways associated with changes in hnRNP K expression. Here, we observed that alterations in hnRNP K expression result in an impairment of ribosomal biogenesis and activation of pathways directly responsible for global translation. Using both knockdown and overexpression systems, we observed a direct correlation between hnRNP K expression and expression of S6, S6K, phosphorylated S6, eIF and mTOR pathways and uncovered defects in rRNA splicing. Collectively, these data indicate that impairment of cap-dependent loading and alterations in ribogenesis may be a driving force in the clinical manifestations of hnRNP K-driven malignancies. Furthermore, these results suggest that translational-inhibitors may be useful in exploiting hnRNP K-dependent vulnerabilities. To examine this aspect, we are currently using FDA-approved translation inhibitors and disruptors of ribogenesis (e.g. homoharringtonineand mTOR-inhibitors) and KTP- compounds, respectively. While these indirect targeting strategies are interesting, our results indicate that hnRNP K also regulates cellular programs outside of translation. Thus, potential therapies that effectively target hnRNP K overexpression will require direct inhibition of its RNA binding functions. To this end, we used several screening assays including fluorescence anisotropy (FA), surface plasmon resonance, SYPRO-orange thermal shift assays, and cell proliferation assays to screen 80,000 small molecule compounds which led to the identification of 9 candidates that disrupt hnRNP K-mRNA interactions and cause cell death in an hnRNP K-dependent manner. Further, cellular thermal shift assays revealed these lead compounds engage hnRNP K within cells and most critically, result in reduced expression of hnRNP K targets in vivo. These candidate compounds as well as potentially more potent structural analogs are currently being evaluated. Collectively, our results demonstrate that the oncogenic functions of hnRNP K are amenable to both indirect therapeutic intervention using FDA-approved agents as well as direct inhibition through newly identified small molecule compounds, signifying that there may be a roadmap to effective therapies for hnRNP K-dependent malignancies. Disclosures No relevant conflicts of interest to declare.

Validation and invalidation of SARS-CoV-2 papain-like protease inhibitors

SARS-CoV-2 encodes two viral cysteine proteases, the main protease (Mpro) and the papain-like protease (PLpro), both of which are validated antiviral drug targets. The PLpro is involved in the cleavage of viral polyproteins as well as immune modulation through removing ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins. Therefore, targeting PLpro might be a two-pronged approach. Several compounds including YM155, cryptotanshinone, tanshinone I, dihydrotanshinone I, tanshinone IIA, SJB2-043, 6-thioguanine, and 6-mercaptopurine were recently identified as SARS-CoV-2 PLpro inhibitors through high-throughput screening. In this study, we aim to validate/invalidate the reported PLpro inhibitors using a combination of PLpro target specific assays including enzymatic FRET assay, thermal shift binding assay (TSA), and the cell based FlipGFP assay. Collectively, our results showed that all compounds tested either did not show binding or led to denaturation of the PLpro in the TSA binding assay, which might explain their weak enzymatic inhibition in the FRET assay. In addition, none of the compounds showed cellular inhibition of PLpro as revealed by the FlipGFP assay. Therefore, more efforts are needed to search for specific and potent SARS-CoV-2 PLpro inhibitors.

Aza-SAHA Derivatives are Selective Histone Deacetylase 10 Chemical Probes That Inhibit Polyamine Deacetylation

We report the first selective chemical probes for histone deacetylase 10 (HDAC10) with unprecedented selectivity over other HDAC isozymes. HDAC10 deacetylates polyamines and has a distinct substrate specificity, making it unique among the 11 zinc-dependent HDAC hydrolases. Taking inspiration from HDAC10 polyamine substrates, we systematically inserted an amino group (“aza-scan”) into the hexyl linker moiety of the approved drug Vorinostat (SAHA). This one atom replacement (C-->N) transformed SAHA from an unselective pan-HDAC inhibitor into a specific HDAC10 inhibitor. Optimization of the aza-SAHA structure yielded DKFZ-748, which has a double-digit nanomolar IC50 against HDAC10 in cells and >500-fold selectivity over the closest relative HDAC6 as well as the Class I enzymes (HDAC1, 2, 3, 8). Potency of our aza-SAHA derivatives is rationalized with HDAC10 co-crystal structures and demonstrated by cellular and biochemical target-engagement, as well as thermal-shift, assays. Treatment of cells with DKFZ-748, followed by quantification of selected polyamines, confirmed for the first time the suspected cellular function of HDAC10 as a poly-amine deacetylase. Selective HDAC10 chemical probes provide a valuable pharmacological tool for target validation and will enable further studies on the enigmatic biology of HDAC10 and acetylated polyamines. HDAC10-selective aza-SAHA derivatives are not cytotoxic, which opens the doors to novel therapeutic applications as immunomodulators or in combination cancer therapy.