Differential Scanning Fluorimetry Assay Research Articles

An in vitro and machine learning framework for quantifying serum albumin binding of per- and polyfluoroalkyl substances

Abstract Per- and polyfluoroalkyl substances (PFAS) are a diverse class of anthropogenic chemicals; many are persistent, bioaccumulative, and mobile in the environment. Worldwide, PFAS bioaccumulation causes serious adverse health impacts, yet the physiochemical determinants of bioaccumulation and toxicity for most PFAS are not well understood, largely due to experimental data deficiencies. As most PFAS are proteinophilic, protein binding is a critical parameter for predicting PFAS bioaccumulation and toxicity. Among these proteins, human serum albumin (HSA) is the predominant blood transport protein for many PFAS. We previously demonstrated the utility of an in vitro differential scanning fluorimetry assay for determining relative HSA binding affinities for 24 PFAS. Here, we report HSA affinities for 65 structurally diverse PFAS from 20 chemical classes. We leverage these experimental data, and chemical/molecular descriptors of PFAS, to build 7 machine learning classifier algorithms and 9 regression algorithms, and evaluate their performance to identify the best predictive binding models. Evaluation of model accuracy revealed that the top performing classifier model, logistic regression, had an AUROC statistic of 0.936. The top performing regression model, support vector regression, had an R2 of 0.854. These top performing models were then used to predict HSA-PFAS binding for chemicals in the EPAPFASINV list of 430 PFAS. These developed in vitro and in silico methodologies represent a high-throughput framework for predicting protein-PFAS binding based on empirical data, and generate directly comparable binding data of potential use in predictive modeling of PFAS bioaccumulation and other toxicokinetic endpoints.

Spiro-Acridine Compound as a Pteridine Reductase 1 Inhibitor: in silico Target Fishing and in vitro Studies.

Among the many neglected tropical diseases, leishmaniasis ranks second in mortality rate and prevalence. In a previous study, acridine derivatives were synthesized and tested for their antileishmanial activity against L. chagasi. The most active compound identified in that study (1) showed a single digit IC50 value against the parasite (1.10 μg/mL), but its macromolecular target remained unknown. Aiming to overcome this limitation, this work exploited inverse virtual screening to identify compound 1's putative molecular mechanism of action. In vitro assays confirmed that compound 1 binds to Leishmania chagasi pteridine reductase 1 (LcPTR1), with moderate affinity (Kd=33,1 μM), according to differential scanning fluorimetry assay. Molecular dynamics simulations confirm the stability of LcPTR1-compound 1 complex, supporting a competitive mechanism of action. Therefore, the workflow presented in this work successfully identified PTR1 as a macromolecular target for compound 1, allowing the designing of novel potent antileishmanial compounds.

Quantitative cross-species comparison of serum albumin binding of per- and polyfluoroalkyl substances from five structural classes.

Per- and polyfluoroalkyl substances (PFAS) are a class of over 8000 chemicals, many of which are persistent, bioaccumulative, and toxic to humans, livestock, and wildlife. Serum protein binding affinity is instrumental in understanding PFAS toxicity, yet experimental binding data is limited to only a few PFAS congeners. Previously, we demonstrated the usefulness of a high-throughput, in vitro differential scanning fluorimetry assay for determination of relative binding affinities of human serum albumin for 24 PFAS congeners from 6 chemical classes. In the current study, we used this assay to comparatively examine differences in human, bovine, porcine, and rat serum albumin binding of 8 structurally informative PFAS congeners from 5 chemical classes. With the exception of the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorooctanol (6:2 FTOH), each PFAS congener bound by human serum albumin was also bound by bovine, porcine, and rat serum albumin. The critical role of the charged functional headgroup in albumin binding was supported by the inability of albumin of each species tested to bind 6:2 FTOH. Significant interspecies differences in serum albumin binding affinities were identified for each of the bound PFAS congeners. Relative to human albumin, perfluoroalkyl carboxylic and sulfonic acids were bound with greater affinity by porcine and rat serum albumin, and the perfluoroalkyl ether acid congener bound with lower affinity to porcine and bovine serum albumin. These comparative affinity data for PFAS binding by serum albumin from human, experimental model, and livestock species reduce critical interspecies uncertainty and improve accuracy of predictive bioaccumulation and toxicity assessments for PFAS.

Structural basis for the development of potential inhibitors targeting FadD23 from Mycobacterium tuberculosis.

Sulfolipid-1 (SL-1) is a lipid that is abundantly found in the cell wall of Mycobacterium tuberculosis (Mtb). MtbFadD23 is crucial in the SL-1 synthesis pathway. Previously, 5'-O-[N-(11-phenoxyundecanoyl)sulfamoyl]adenosine (PhU-AMS) has been shown to be a general inhibitor of fatty-acid-adenylating enzymes (FadDs) in Mtb. However, the fatty acyl-AMP ligase (FAAL) class of FadDs, which includes MtbFadD23, appears to be functionally nonredundant in the production of multiple fatty acids. In this study, the ability of PhU-AMS to bind to MtbFadD23 was examined under in vitro conditions. The crystal structure of the MtbFadD23-PhU-AMS complex was determined at a resolution of 2.64 Å. Novel features were identified by structural analysis and comparison. Although PhU-AMS could bind to MtbFadD23, it did not inhibit the FAAL adenylation activity of MtbFadD23. However, PhU-AMS improved the main Tm value in a differential scanning fluorimetry assay, and a structural comparison of MtbFadD23-PhU-AMS with FadD32 and PA1221 suggested that PhU-AMS blocks the loading of the acyl chain onto Pks2. This study sheds light on the structure-based design of specific inhibitors of MtbFadD23 and general inhibitors of FAALs.

Design, synthesis, and biological screening of a series of pyrazolo [1,5-a]quina-zoline derivatives as SIRT6 activators

SIRT6 has emerged as a novel therapeutic target for a variety of diseases. In this study, a total of 102 pyrazolo [1,5-a]quinazoline derivatives were designed and synthesized. The result revealed that 2-methyl-N-(4-phenoxy-phenyl)pyrazolo [1,5-a]quinazoline-5-amine (21q) was the most active compound by structure-activity relationship study, which significantly enhanced SIRT6 defatty-acylation activity with an EC1.5 value of 1.85±0.41 μM and EC50 value of 11.15±0.33 μM. The biological activity of 21q was further verified by differential scanning fluorimetry assay (DSF) and surface plasmon resonance assay (SPR). Molecular docking showed that the pyrazolo [1,5-a]quinazoline of 21q formed a hydrogen bond with Val115 and four π- π interactions with Phe64, Phe82 and Phe86. 21q can significantly improve the thermal stability of SIRT6 protein and inhibit the PI3K/Akt signaling pathway in mouse embryonic fibroblasts (MEFs), thereby inhibiting the proliferation of MEFs. Collectively, we discovered a new potent SIRT6 activator, which can be taken as a lead compound for later studies.

Application of temperature-responsive HIS-tag fluorophores to differential scanning fluorimetry screening of small molecule libraries.

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient, most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in histidine-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this HIS-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

Rapid Characterization of Human Serum Albumin Binding for Per- and Polyfluoroalkyl Substances Using Differential Scanning Fluorimetry.

Per- and polyfluoroalkyl substances (PFAS) are a diverse class of synthetic chemicals that accumulate in the environment. Many proteins, including the primary human serum transport protein albumin (HSA), bind PFAS. The predictive power of physiologically based pharmacokinetic modeling approaches is currently limited by a lack of experimental data defining albumin-binding properties for most PFAS. A novel thermal denaturation assay was optimized to evaluate changes in the thermal stability of HSA in the presence of increasing concentrations of known ligands and a structurally diverse set of PFAS. Assay performance was initially evaluated for fatty acids and HSA-binding drugs ibuprofen and warfarin. Concentration-response relationships were determined and dissociation constants (Kd) for each compound were calculated using regression analysis of the dose-dependent changes in HSA melting temperature. Estimated Kd values for HSA binding of octanoic acid, decanoic acid, hexadecenoic acid, ibuprofen, and warfarin agreed with established values. The binding affinities for 24 PFAS that included perfluoroalkyl carboxylic acids (C4-C12), perfluoroalkyl sulfonic acids (C4-C8), mono- and polyether perfluoroalkyl ether acids, and polyfluoroalkyl fluorotelomer substances were determined. These results demonstrate the utility of this differential scanning fluorimetry assay as a rapid high-throughput approach for determining the relative protein-binding properties and identification of chemical structures involved in binding for large numbers of structurally diverse PFAS.

Abstract 4520: Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice

Abstract Introduction: The STING (STimulator of INterferon Genes) protein has been identified as an attractive target for cancer immunotherapy due to its central role in inducing T-cell mediated tumor control. Here we describe the preclinical characteristics of the novel STING agonist, ALG-031048, that demonstrates potent anti-tumoral activity in in vivo mouse models. Methods: Binding to STING was determined using differential scanning fluorimetry (DSF). Cellular activation and cytokine release were assessed using HEK 293T reporter cell lines as well as THP-1 and Raw264.7 cells. In vitro stability was measured in the presence of snake venom phosphodiesterase (SVPD). In vivo anti-tumoral activity was tested in the syngeneic CT26 and B16F10 mouse model. Results: ALG-031048 demonstrated potent binding to the STING R232 protein in the DSF assay with a mean Kd of 3.12 ± 0.12 μM and a thermal shift of 12.8 °C, similar to the natural ligand 2'3'cGAMP (Kd of 2.75 ± 0.78 μM, shift of 16.7°C) or the clinical stage STING agonist, ADU-S100 (Kd of 4.61 ± 0.42 μM, shift of 10.7°C). In the HEK 293T R232 reporter assay, ALG-031048 was more potent than ADU-S100 with a mean EC50 of 55.9 ± 24.1 and 30.3 ± 17.1 nM for the IFNβ and IRF reporter, respectively, compared to ADU-S100 with EC50 values of 233 ± 179 and 88.7 ± 35.6 nM. Cellular activation was confirmed in THP-1 and Raw264.7 cells where ALG-031048 induced the secretion of IFNβ and CXCL-10. ALG-031048 was highly stable and exhibited no degradation for up to 24 hours in the presence of SVPD while ADU-S100 and 2'3'cGAMP were quickly degraded with less than 10% remaining after 24 hours. In vivo, 3 intra-tumoral (IT) doses of 100 μg ALG-031048 q3d caused complete tumor regression in 90% of animals in the CT26 mouse colon carcinoma model, while treatment with 25 μg ALG-031048 IT 3xq3d resulted in survival of 60 % of animals. In contrast, only 10 and 44% of mice showed complete tumor regression when treated IT 3xq3d with 25 or 100 μg ADU-S100, respectively. To study whether induction of a protective immune response was associated with STING agonist treatment, surviving animals were re-challenged with a second inoculation of CT26 cells on the contra-lateral side. All naïve animals formed tumors within 23-30 days. In contrast, 5 out of 5 and 8 out of 9 mice pre-treated with 25 or 100 μg ALG-031048, respectively, did not exhibit any significant tumor growth over 40 days, while 3 out of 4 animals pretreated with 100 μg ADU-S100 showed complete tumor suppression. Initial results using the B16F10 mouse melanoma model confirmed the potent anti-tumoral activity of ALG-031048 observed in the CT26 model. Summary: ALG-031048 is a novel STING agonist combining high stability and excellent in vitro potency. In preclinical models, ALG-031048 demonstrated potent and long-lasting tumor suppression. ALG-031048 is advancing in toxicology studies for further profiling as a clinical candidate. Citation Format: Andreas Jekle, Santosh Thatikonda, Sarah Stevens, Caroline Williams, April Kinkade, Suping Ren, Ruchika Jaisinghani, Qingling Zhang, Dinah Misner, Antitsa Stoycheva, Jerome Deval, Sucheta Mukherjee, Francois Gonzalvez, Sushmita Chanda, David B. Smith, Julian A. Symons, Lawrence M. Blatt, Leonid Beigelman. Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4520.

Rapid Screening of Additive Formulations for Enhancing Xylanase Stability in Pulp Bleaching and Storage Conditions

Enzyme deactivation is a significant parameter to restrain the industrial commercialization of enzymes. In this study, a high-throughput screening technique using differential scanning fluorimetry (DSF) incorporated with enzyme activity analysis was thus developed in order to identify a suitable additive formulation for promoting enzyme stability in terms of conformational stability and functional activity. To demonstrate the concept feasibility, endo-xylanase obtained from metagenome of termite gut-inhabiting bacteria (XYN12) was used as a study model and several group of additives including salts, polymers, polyols and sugars at various concentrations and different combination patterns (single, double and triple pairing) were applied for investigating their effects on enzyme stability. As a result, a total of 80 reaction mixtures were retrieved including a native control (enzyme without any additives). DSF assay was performed using real-time PCR instrument to detect unfolding of protein in the presence of a florescence dye. Accurate melting temperature (Tm) values of each enzyme formulation were calculated using Boltzmann equation and compared to the Tm of the native control in order to find ?Tm. After that, the residual activity of xylanase in all reaction mixtures were measured using pHBAH method in automated liquid handling system. For single additives, glycerol had a stabilizing effect to the target enzyme, as observed by a significant increase in the ?Tm, while ?(NH4)2SO4, CaCl2, NaCl destabilized it. In addition, cocktail additives included glycerol, PEG4000 and trehalose dramatically enhanced the enzyme stability by positive shifting of Tm from 54 to 56 (C and increasing of relative activity by more than 4 folds compared to single additive. Storage stability of the enzyme was improved when mixed with this cocktail additive as indicated by remaining of the enzyme activity (86 %) after kept at 30 (C, pH 7.4 for 262 days, whereas native control lost almost of its activity within 40 days when stored at the same condition.

Treatment of an aggressive orthotopic murine glioblastoma model with combination checkpoint blockade and a multivalent neoantigen vaccine.

Although clinical trials testing immunotherapies in glioblastoma (GBM) have yielded mixed results, new strategies targeting tumor-specific somatic coding mutations, termed "neoantigens," represent promising therapeutic approaches. We characterized the microenvironment and neoantigen landscape of the aggressive CT2A GBM model in order to develop a platform to test combination checkpoint blockade and neoantigen vaccination. Flow cytometric analysis was performed on intracranial CT2A and GL261 tumor-infiltrating lymphocytes (TILs). Whole-exome DNA and RNA sequencing of the CT2A murine GBM was employed to identify expressed, somatic mutations. Predicted neoantigens were identified using the pVAC-seq software suite, and top-ranking candidates were screened for reactivity by interferon-gamma enzyme linked immunospot assays. Survival analysis was performed comparing neoantigen vaccination, anti-programmed cell death ligand 1 (αPD-L1), or combination therapy. Compared with the GL261 model, CT2A exhibited immunologic features consistent with human GBM including reduced αPD-L1 sensitivity and hypofunctional TILs. Of the 29 CT2A neoantigens screened, we identified neoantigen-specific CD8+ T-cell responses in the intracranial TIL and draining lymph nodes to two H2-Kb restricted (Epb4H471L and Pomgnt1R497L) and one H2-Db restricted neoantigen (Plin2G332R). Survival analysis showed that therapeutic neoantigen vaccination with Epb4H471L, Pomgnt1R497L, and Plin2G332R, in combination with αPD-L1 treatment was superior to αPD-L1 alone. We identified endogenous neoantigen specific CD8+ T cells within an αPD-L1 resistant murine GBM and show that neoantigen vaccination significantly augments survival benefit in combination with αPD-L1 treatment. These observations provide important preclinical correlates for GBM immunotherapy trials and support further investigation into the effects of multimodal immunotherapeutic interventions on antiglioma immunity. 1. Neoantigen vaccines combined with checkpoint blockade may be promising treatments.2. CT2A tumors exhibit features of human GBM microenvironments.3. Differential scanning fluorimetry assays may complement in silico neoantigen prediction tools.

Lessons from LIMK1 enzymology and their impact on inhibitor design.

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique ‘rock-and-poke’ mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding.

The Natural Product Butylcycloheptyl Prodiginine Binds Pre-miR-21, Inhibits Dicer-Mediated Processing of Pre-miR-21, and Blocks Cellular Proliferation

Identification of RNA-interacting pharmacophores could provide chemical probes and, potentially, small molecules for RNA-based therapeutics. Using a high-throughput differential scanning fluorimetry assay, we identified small-molecule natural products with the capacity to bind the discrete stem-looped structure of pre-miR-21. The most potent compound identified was a prodiginine-type compound, butylcycloheptyl prodiginine (bPGN), with the ability to inhibit Dicer-mediated processing of pre-miR-21 invitro and in cells. Time-dependent RT-qPCR, western blot, and transcriptomic analyses showed modulation of miR-21 expression and its target genes such as PDCD4 and PTEN upon treatment with bPGN, supporting on-target inhibition. Consequently, inhibition of cellular proliferation in HCT-116 colorectal cancer cells was also observed when treated with bPGN. The discovery that bPGN can bind and modulate the expression of regulatory RNAs such as miR-21 helps set the stage for further development of this class of natural product as a molecular probe or therapeutic agent against miRNA-dependent diseases.

Fragment-based screening identifies inhibitors of ATPase activity and of hexamer formation of Cag\u03b1 from the Helicobacter pylori type IV secretion system

Type IV secretion systems are multiprotein complexes that mediate the translocation of macromolecules across the bacterial cell envelope. In Helicobacter pylori a type IV secretion system encoded by the cag pathogenicity island encodes 27 proteins and most are essential for virulence. We here present the identification and characterization of inhibitors of Cagα, a hexameric ATPase and member of the family of VirB11-like proteins that is essential for translocation of the CagA cytotoxin into mammalian cells. We conducted fragment-based screening using a differential scanning fluorimetry assay and identified 16 molecules that stabilize the protein suggesting that they bind Cagα. Several molecules affect binding of ADP and four of them inhibit the ATPase activity. Analysis of enzyme kinetics suggests that their mode of action is non-competitive, suggesting that they do not bind to the active site. Cross-linking suggests that the active molecules change protein conformation and gel filtration and transmission electron microscopy show that molecule 1G2 dissociates the Cagα hexamer. Addition of the molecule 1G2 inhibits the induction of interleukin-8 production in gastric cancer cells after co-incubation with H. pylori suggesting that it inhibits Cagα in vivo. Our results reveal a novel mechanism for the inhibition of the ATPase activity of VirB11-like proteins.

Natural products as inhibitors of Leishmania major dihydroorotate dehydrogenase

The flavoenzyme dihydroorotate dehydrogenase (DHODH) catalyzes the fourth reaction of the de novo pyrimidine biosynthetic pathway, which exerts vital functions in the cells, especially within DNA and RNA biosynthesis. Thus, this enzyme stands out as a new key molecular target for parasites causing Neglected Diseases (NDs). Focused on contributing to the development of new therapeutic alternatives for NDs, in this study, for the first time, a screening of 57 natural products for in vitro inhibition of Leishmania major DHODH (LmDHODH) was carried out, including cross validation against the human DHODH (HsDHODH). A subset of natural products consisting of 21 sesquiterpene lactones (STLs) was submitted to QSAR studies. Additionally, thermostability studies by differential scanning fluorimetry (DSF) were performed to determine whether the STLs are effectively or not binding to the enzyme. The IC50 values against LmDHODH varied from 27 to 1200 μM; only irrelevant inhibition was obtained on HsDHODH. DSF assays confirmed binding of STLs to LmDHODH; moreover, it is suggested that such inhibitors might act in a different site other than the active site. A reliable QSAR model based on molecular descriptors was obtained (R2: 0.83; Q2CV: 0.69 and Q2EXT/F2: 0.66) indicating that stronger inhibition requires a balanced distribution of the hydrophobic regions across the molecular surface, as well as higher width and lower hydrophobicity of the molecules. A pharmacophore-based 3D-QSAR approach also afforded a useful model (R2: 0.72; Q2CV: 0.50 and Q2EXT/F2: 0.62), which confirmed the importance of proper orientation of the ligands, molecular surface features and shape for stronger inhibition, reflecting properties of a putative common binding site. These data indicated for the first time that natural products can actually inhibit LmDHODH and highlighted some metabolites as potentially interesting starting points for the discovery of more potent LmDHODH inhibitors, ultimately aiming at new effective therapeutic alternatives for leishmaniasis and, possibly, other NDs caused by trypanosomatids.

An allelic series at the KARRIKIN INSENSITIVE2 locus of Arabidopsis thaliana decouples ligand hydrolysis and receptor degradation from downstream signalling.

Karrikins are butenolide compounds present in post-fire environments that can stimulate seed germination in many species, including Arabidopsis thaliana. Plants also produce endogenous butenolide compounds that serve as hormones, namely strigolactones (SLs). The receptor for karrikins (KARRIKIN INSENSITIVE2; KAI2) and the receptor for SLs (DWARF14; D14) are homologous proteins that share many similarities. The mode of action of D14 as a dual enzyme receptor protein is well established, but the nature of KAI2-dependent signalling and its function as a receptor are not fully understood. To expand our knowledge of how KAI2 operates, we screened ethyl methanesulphonate (EMS)-mutagenized populations of A.thaliana for mutants with kai2-like phenotypes and isolated 13 new kai2 alleles. Among these alleles, kai2-10 encoded a D184N protein variant that was stable inplanta. Differential scanning fluorimetry assays indicated that the KAI2 D184N protein could interact normally with bioactive ligands. We developed a KAI2-active version of the fluorescent strigolactone analogue Yoshimulactone Green to show that KAI2 D184N exhibits normal rates of ligand hydrolysis. KAI2 D184N degraded in response to treatment with exogenous ligands, suggesting that receptor degradation is a consequence of ligand binding and hydrolysis, but is insufficient for signalling activity. Remarkably, KAI2 D184N degradation was hypersensitive to karrikins, but showed a normal response to strigolactone analogues, implying that these butenolides may interact differently with KAI2. These results demonstrate that the enzymatic and signalling functions of KAI2 can be decoupled, and provide important insights into the mechanistic events that underpin butenolide signalling in plants.

Substrate interaction defects in histidyl-tRNA synthetase linked to dominant axonal peripheral neuropathy.

Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.

Retention of the Structure and Function of Heparan Sulfate Biomaterials After Gamma Irradiation.

Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as "HS3") is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range of diseases and disorders.

Inhibition studies of DNA methyltransferases by maleimide derivatives of RG108 as non-nucleoside inhibitors.

DNA methyltransferases (DNMTs) are important drug targets for epigenetic therapy of cancer. Nowadays, non-nucleoside DNMT inhibitors are in development to address high toxicity of nucleoside analogs. However, these compounds still have low activity in cancer cells and mode of action of these compounds remains unclear. In this work, we studied maleimide derivatives of RG108 by biochemical, structural and computational approaches to highlight their inhibition mechanism on DNMTs. Findings demonstrated a correlation between cytotoxicity on mesothelioma cells of these compounds and their inhibitory potency against DNMTs. Noncovalent and covalent docking studies, supported by crystallographic (apo structure of M.HhaI) and differential scanning fluorimetry assays, provided detailed insights into their mode of action and revealed essential residues for the stabilization of such compounds inside DNMTs. [Formula: see text].

Novel In Vitro Screening System Based on Differential Scanning Fluorimetry to Search for Small Molecules against the Disassembly or Assembly of HIV-1 Capsid Protein.

Varieties of in vitro systems have been used to study biochemical properties of human immunodeficiency virus Gag-capsid protein (HIV Gag-CA). Recently, we have comparatively characterized HIV-1 and HIV-2 Gag-CA proteins using such technology, and have demonstrated that the NaCl-initiated CA-polymerization in vitro and the stability of CA N-terminal domain as judged by differential scanning fluorimetry (DSF) are inversely correlated. In this study, we found that ZnCl2 works as a competent initiator of the in vitro HIV-1 CA-polymerization at much lower concentrations than those of NaCl frequently used for the polymerization initiation. We also showed by DSF assays that ZnCl2 highly destabilize HIV-1 CA. Furthermore, PF74, a well-known inducer of premature HIV-1 uncoating in infected cells, was demonstrated to unusually promote the HIV-1 CA-disassembly in the presence of ZnCl2 as revealed by DSF assays. Taken together, we conclude that the DSF method may be useful as an efficient monitoring system to screen anti-HIV-1 CA molecules.

Extraction of Protein Thermodynamic Parameters from High‐Throughput Differential Scanning Fluorimetry Assays

Thermodynamic properties of protein unfolding have been extensively studied; however, the methods used have typically required significant preparation time and high protein concentrations. Here, we present a facile, simple, and high‐throughput differential scanning fluorimetry (DSF) method that enables a complete set of thermodynamic parameters of protein unfolding to be extracted. This method assumes a two‐state, reversible protein unfolding mechanism and provides the capacity to quickly analyze the biophysical mechanisms of changes in protein stability and to more thoroughly characterize the effect of additives, inhibitors, or pH. We show the utility of the DSF method by analyzing the thermal denaturation of lysozyme, carbonic anhydrase, chymotrypsin, horseradish peroxidase, and cellulase. Compared to similar biophysical analyses by circular dichroism, DSF provides greater than 24‐fold reduction in experimental time. This study opens to door to rapid characterization of protein stability on low concentration protein samples.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP).