Differential Scanning Fluorescence Research Articles

1,3,4-oxadiazoles as inhibitors of the atypical member of the BET family bromodomain factor 3 from Trypanosoma cruzi (TcBDF3).

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions globally, with increasing urban cases outside of Latin America. Treatment is based on two compounds, namely, benznidazole (BZ) and nifurtimox, but chronic cases pose several challenges. Targeting lysine acetylation, particularly bromodomain-containing proteins, shows promise as a novel antiparasitic target. Our research focuses on TcBDF3, a cytoplasmic protein, which is crucial for parasite differentiation that recognizes acetylated alpha-tubulin. In our previous study, A1B4 was identified as a high-affinity binder of TcBDF3, showing significant trypanocidal activity with low host toxicity in vitro. In this report, the binding of TcBDF3 to A1B4 was validated using differential scanning fluorescence, fluorescence polarization, and molecular modeling, confirming its specific interaction. Additionally, two new 1,3,4-oxadiazoles derived from A1B4 were identified, which exhibited improved trypanocide activity and cytotoxicity profiles. Furthermore, TcBDF3 was classified for the first time as an atypical divergent member of the bromodomain extraterminal family found in protists and plants. These results make TcBDF3 a unique target due to its localization and known functions not shared with higher eukaryotes, which holds promise for Chagas disease treatment.

Detection of CD40 Protein-Umbelliferone Interaction via Differential Scanning Fluorescence.

The investigation of interactions between different molecules is a crucial aspect of understanding disease pathogenesis and screening for drug targets. Umbelliferone, an active ingredient in Tibetan medicine Vicatia thibetica, exhibits an immunomodulatory effect with an unknown mechanism. The CD40 protein is a key target in the immune response. Therefore, this study employs the principle of differential scanning fluorescence technology to analyze the interactions between CD40 protein and umbelliferone using fluorescent enzyme markers. Initially, the stability of the protein fluorescent orange dye was experimentally verified, and the optimal dilution ratio of 1:500 was determined. Subsequently, it was observed that the temperature melting (Tm) value of CD40 protein tended to decrease with an increase in concentration. Interestingly, the interaction between CD40 protein and umbelliferone was found to enhance the thermal stability of CD40 protein. This study represents the first attempt to detect the binding potential of small molecule compounds and proteins using fluorescence microplates and fluorescent dyes. The technique is characterized by high sensitivity and accuracy, promising advancements in the fields of protein stability, protein structure, and protein-ligand interactions, thus facilitating further research and exploration.

Screening approaches for the identification of Nrf2-Keap1 protein-protein interaction inhibitors targeting hot spot residues

Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.

Basement Membrane Mimetic Hydrogel Cooperates with Rho-Associated Protein Kinase Inhibitor to Promote the Development of Acini-Like Salivary Gland Spheroids.

Successful engineering of functional salivary glands necessitates the creation of cell-instructive environments for ex vivo expansion and lineage specification of primary human salivary gland stem cells (hS/PCs). Herein, basement membrane mimetic hydrogels were prepared using hyaluronic acid, cell adhesive peptides, and hyperbranched polyglycerol (HPG), with or without sulfate groups, to produce "hyperGel+" or "hyperGel", respectively. Differential scanning fluorescence experiments confirmed the ability of the sulphated HPG precursor to stabilize fibroblast growth factor 10. The hydrogels were nanoporous, cytocompatibile and cell-permissive, enabling the development of multicellular hS/PC spheroids in 14 days. Incorporation of sulfated HPG species in the hydrogel enhanced cell proliferation. Culture of hS/PCs in hyperGel+ in the presence of a Rho kinase inhibitor, Y-27632 (Y-27), led to the development of spheroids with a central lumen, increased the expression of acinar marker aquaporin-3 at the transcript level (AQP3), and decreased the expression of ductal marker keratin 7 at both the transcript (KRT7) and the protein levels (K7). Reduced expression of transforming growth factor beta (TGF-β) targets SMAD2/3 was also observed in Y27-treated cultures, suggesting attenuation of TGF-β signaling. Thus, hyperGel+ cooperates with the ROCK inhibitor to promote the development of lumened spheroids with enhanced expression of acinar markers.

Structure-Activity Relationship of Novel Pyrimidine Derivatives with Potent Inhibitory Activities against Mycobacterium tuberculosis.

Discovery of novel antitubercular drugs is an effective strategy against drug-resistant tuberculosis (TB). Our previous study has identified LPX-16j as a novel antitubercular compound. Herein, we perform a comprehensive structure-activity relationship (SAR) based on LPX-16j, indicating that the central pyrimidine ring moiety was crucial for the antitubercular activities of its derivatives, and replacing the naphthyl group with hydrophobic substitutes was well tolerated. The representative derivative 5a exhibited potent activity against H37Ra, H37Rv, and clinical drug-resistant TB with minimum inhibitory concentration (MIC) values of 0.5-1.0 μg/mL. Meanwhile, 5a showed an acceptable safety in vivo and displayed a favorable oral bioavailability with a value of 40.7%. The differential scanning fluorescence, isothermal titration calorimetry, and molecular docking assays indicated that PknB could be one of the targets of compound 5a. Overall, this study identified 5a as a novel promising lead compound with the potential to develop candidates for the treatment of drug-resistant TB.

Peptide transport in Bacillus subtilis - structure and specificity in the extracellular solute binding proteins OppA and DppE.

Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake - the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with K d values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes.

A structure-function study of ZraP and ZraS provides new insights into the two-component system Zra

BackgroundZra belongs to the envelope stress response (ESR) two-component systems (TCS). It is atypical because of its third periplasmic repressor partner (ZraP), in addition to its histidine kinase sensor protein (ZraS) and its response regulator (ZraR) components. Furthermore, although it is activated by Zn2+, it is not involved in zinc homeostasis or protection against zinc toxicity. Here, we mainly focus on ZraS but also provide information on ZraP. MethodsThe purified periplasmic domain of ZraS and ZraP were characterized using biophysical and biochemical technics: multi-angle laser light scattering (MALLS), circular dichroism (CD), differential scanning fluorescence (DSF), inductively coupled plasma atomic emission spectroscopy (ICP-AES), cross-linking and small-angle X-ray scattering (SAXS). In-vivo experiments were carried out to determine the redox state of the cysteine residue in ZraP and the consequences for the cell of an over-activation of the Zra system. ResultsWe show that ZraS binds one Zn2+ molecule with high affinity resulting in conformational changes of the periplasmic domain, consistent with a triggering function of the metal ion. We also demonstrate that, in the periplasm, the only cysteine residue of ZraP is at least partially reduced. Using SAXS, we conclude that the previously determined X-ray structure is different from the structure in solution. ConclusionOur results allow us to propose a general mechanism for the Zra system activation and to compare it to the homologous Cpx system. General significanceWe bring new input on the so far poorly described Zra system and notably on ZraS.

Abstract 3752: Identification of nanobodies specific for the oncogenic protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) that modulate its binding, stability, and phosphatase activity

Abstract PRL-3 is an oncogenic phosphatase across multiple cancer types, including colon, ovarian, melanoma, breast, and leukemia. While there is increasing interest in developing PRL-3 inhibitors for use in cancer research and treatment, there have been several issues in designing small molecule inhibitors for PRL-3, such as a shallow, negative charged active site, homology between the PRL-3 active site and other protein tyrosine phosphatases, and a high degree of overall homology between PRL-3 and family members PRL-1 and PRL-2. Nanobodies have recently emerged as an immensely useful research tool and show promise as a cancer therapeutic. Nanobodies are small, at ~15kD and lack light chains, allowing them to fit into spaces on target proteins that conventional antibodies cannot normally reach. Other advantages of nanobodies include their stability under stringent conditions, lack of immunogenicity, ability to permeate the cell, and a high specificity and affinity for their antigens. Using full-length PRL-3 protein as an immunogen in alpaca, I used phage display technology to identify alpaca nanobodies that had high affinity for PRL-3 through subtractive panning. I identified 18 unique nanobodies through sequencing; 14 of these were able to be expressed in bacteria and purified. The binding specificity of the nanobodies to PRL-3 over PRL-1 and PRL-2 was determined through an indirect ELISA assay, which showed that that 12 out of 14 anti-PRL-3 nanobodies bound PRL-3 significantly better than PRL-1 or PRL-2 (~25X times higher binding affinity to PRL-3, p<0.0001). I also identified several nanobodies that stabilize PRL-3 structure using a Differential Scanning Fluorescence (DSF) assays to analyze shifts in the melting temperature of PRL-3 following binding, with the ultimate goal of developing PRL-3/nanobody crystal structures to identify nanobody binding sites and find PRL-3 active site binders. Additionally, I have found the 6X-Histidine-tagged nanobodies are useful for PRL-3 western blot, immunoprecipitation, and immunofluorescence, and mCherry-tagged nanobodies (chromobodies) allow for analysis of PRL-3 trafficking. Nanobodies were also tested for their ability to inhibit the phosphatase activity of PRL-3, using both purified protein and functional in vitro assays. Preliminary results showed that several nanobodies decreased PRL-3 phosphatase activity, and cell-based assays to assess how nanobody expression affects PRL-3 function and cellular phenotype are ongoing. Overall, we have developed. both a novel research tool that can be used to gain insight into the structure and function of PRL-3 in normal and cancer cells, and a potentially new biologic inhibitor of PRL-3 that functions with high specificity and potency. Citation Format: Caroline N. Smith, K. Martin Chow, Louis B. Hersh, Jessica S. Blackburn. Identification of nanobodies specific for the oncogenic protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) that modulate its binding, stability, and phosphatase activity [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 3752.

Characterization and stabilization in process development and product formulation for super large proteinaceous particles.

Super large proteinaceous particles (SLPPs) such as virus, virus like particles, and extracellular vesicles have successful and promising applications in vaccination, gene therapy, and cancer treatment. The unstable nature, the complex particulate structure and composition are challenges for their manufacturing and applications. Rational design of the processing should be built on the basis of fully understanding the characteristics of these bio‐particles. This review highlights useful analytical techniques for characterization and stabilization of SLPPs in the process development and product formulations, including high performance size exclusion chromatography, multi‐angle laser light scattering, asymmetrical flow field‐flow fractionation, nanoparticle tracking analysis, CZE, differential scanning calorimetry, differential scanning fluorescence, isothermal titration calorimetry , and dual polarization interferometry. These advanced analytical techniques will be helpful in obtaining deep insight into the mechanism related to denaturation of SLPPs, and more importantly, in seeking solutions to preserve their biological functions against deactivation or denaturation. Combination of different physicochemical techniques, and correlation with in vitro or in vivo biological activity analyses, are considered to be the future trend of development in order to guarantee a high quality, safety, and efficacy of SLPPs.

Development and optimization of a cascade of screening assays for inhibitors of TRF2.

TRF2 is a telomere associated protein which plays an important role in telomere maintenance. Knockdown of TRF2 can cause chromosomal end to end fusions and induce DNA damage responses. TRF2 exerts its functions partially by recruiting a number of accessory proteins through its TRF homology domain (TRFH), therefore identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions of TRF2 with its associated proteins is important to elucidate the molecular mechanism of these protein-protein interactions. Development of robust and sensitive screening and evaluation assays is critical to the identification of TRF2 inhibitors, in this paper we reported the development and optimization of a cascade of screening and binding affinity evaluation assays, including a competitive FP (Fluorescence Polarization) assay utilized in our previous research, and two novel label-free DSF (Differential Scanning Fluorescence) and BLI (Biolayer Interferometry) assays. A previously identified TRF2 inhibitor TRF2-27 was used as an internal reference compound and evaluated in all of these assays. According to the results, DSF assay is not suitable for TRF2 screening because of the low ΔTm, while the optimized labeled-free BLI assay was demonstrated to be an accurate and reproducible assay for TRF2 inhibitor screening and characterization.

Mechanistic Studies of Cytochrome P450 3A4 Time-Dependent Inhibition Using Two Cysteine-Targeting Electrophiles

Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.

New role of the antidepressant imipramine as a Fascin1 inhibitor in colorectal cancer cells

Serrated adenocarcinoma (SAC) is more invasive, has worse outcomes than conventional colorectal carcinoma (CRC), and is characterized by frequent resistance to anti-epidermal growth factor receptor (EGFR) and overexpression of fascin1, a key protein in actin bundling that plays a causative role in tumor invasion and is overexpressed in different cancer types with poor prognosis. In silico screening of 9591 compounds, including 2037 approved by the Food and Drug Administration (FDA), was performed, and selected compounds were analyzed for their fascin1 binding affinity by differential scanning fluorescence. The results were compared with migrastatin as a typical fascin1 inhibitor. In silico screening and differential scanning fluorescence yielded the FDA-approved antidepressant imipramine as the most evident potential fascin1 blocker. Biophysical and different in vitro actin-bundling assays confirm this activity. Subsequent assays investigating lamellipodia formation and migration and invasion of colorectal cancer cells in vitro using 3D human tissue demonstrated anti-fascin1 and anti-invasive activities of imipramine. Furthermore, expression profiling suggests the activity of imipramine on the actin cytoskeleton. Moreover, in vivo studies using a zebrafish invasion model showed that imipramine is tolerated, its anti-invasive and antimetastatic activities are dose-dependent, and it is associated with both constitutive and induced fascin1 expression. This is the first study that demonstrates an antitumoral role of imipramine as a fascin1 inhibitor and constitutes a foundation for a molecular targeted therapy for SAC and other fascin1-overexpressing tumors.

Mutation in the VP2 gene of P1-2A capsid protein increases the thermostability of virus-like particles of foot-and-mouth disease virus serotype O.

Foot-and-mouth disease (FMD) is an economically important, global disease of cloven-hoofed animals. The conventional vaccine could bring down the incidence of disease in many parts of the world but has many limitations and in India, the disease is enzootic. More promisingly, the alternate vaccine candidates, virus-like particles (VLPs) are as immunogenic as a native virus but are more labile to heat than the live virus capsids. To produce stable VLPs, a single amino acid residue was mutated at 93 and 98 positions at VP2 inter-pentamer region of the P1-2A gene of FMD virus serotype O (IND/R2/75). The mutated capsid protein was expressed in insect cells and characterized for temperature and varying pH stability. Out of S93Y, S93F, S93C, S93H, and Y98F mutant, VLPs, S93Y, S93F, and Y98F showed improved stability at 37°C for 75days compared to wild capsid, which was evaluated by sandwich ELISA. Further, the stability analysis of purified VLPs either by differential scanning fluorescence (DSF) stability assay at different temperatures and pH conditions or by dissociation kinetics showed that the Y98F mutant VLPs were more stable than S93Y, S93F, S93C, and S93H mutant and wild-type VLPs. Immunization of guinea pigs with Y98F VLPs induced neutralizing antibodies and 60% of the animals were protected from the FMDV "O" 100 GPID50 challenge virus.

Utility of High Throughput Screening Techniques to Predict Stability of Monoclonal Antibody Formulations During Early Stage Development

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development. The utility of using accelerated stability compared to HTS techniques (differential scanning light scattering and differential scanning fluorescence) for early formulation screening was evaluated along with the impact of excipients of various types on aggregation of monoclonal antibodies from multiple IgG subtypes. The excipient rank order using quantitative HTS measures was found to correlate with accelerated stability aggregation rate ranking for only 33% (by differential scanning fluorescence) to 42% (by differential scanning light scattering) of the antibodies tested, due to the high intrinsic stability and minimal impact of excipients on aggregation rates and HTS data. Also explored was a case study of employing a platform formulation instead of broader formulation screening for early formulation development.

88. Structural Studies Into the Stability of Novel In Silico Reconstructed Ancestral AAV Vectors

Adeno-associated virus (AAV) vectors have emerged as therapy preferred clinical in vivo gene transfer system. Yet, their sensitivity to pre-existing immunity (PEI) and low carrying capacity remain major drawbacks that limit broad applications. We previously hypothesized and have shown that ancestral species of AAV, which we inferred using statistical and bio-informatic approaches, and generated synthetically, have reduced susceptibility to PEI. Here, our ambition was to study from a biophysical and structural perspective the evolutionary process of AAV using these novel reagents, and to develop correlates of those measures with virus and vector phenotypes.By the means of polyacrylamide gel electrophoresis, intrinsic fluorescence spectroscopy and differential scanning fluorescence, we derive the thermostability profiles of 6 contemporary AAV serotypes (AAV1, AAV2, AAV5, AAV8, AAV9, rh32.33) and 3 ancestral AAV serotypes, called Anc80, Anc81 and Anc82 and differing from AAV8 in VP1 protein sequence by 9.3%, 7.8% and 5.3%, respectively. A theoretical structure of Anc80L0065 VP3 monomer was derived by homology modeling and compared to the crystal structures of AAV2, AAV8 and rh32.33.Structural modeling indicates significant sequence and structure differences between ancestral and contemporary VP3 protein subunits in loops I, IV, VII and VIII, regions that were shown to be involved in antibody and cell receptor binding. Surprisingly, Anc80, Anc81 and Anc82 showed an increase in capsomer melting temperature of approximately 20°C in comparison to AAV8, while vector genome is accessible at lower temperatures. Evolutionary intermediates between Anc80 and AAV8 demonstrate unique melting properties that permit the identification of a limited set of capsid determinants to the thermodynamic stability of AAVs. Our reconstructed phylogeny of AAVs emerges as a relevant tool for sequence-function analysis of AAV vectors.

The N-terminal cytoplasmic region of NCBE displays features of an intrinsic disordered structure and represents a novel target for specific drug screening

The sodium dependent bicarbonate transporter NCBE/NBCn2 is predominantly expressed in the central nervous system (CNS). The highest protein concentrations are found in the choroid plexus. The primary function of this integral plasma membrane transport protein is to regulate intracellular neuronal pH and also probably to maintain the pH homeostasis across the blood-cerebrospinal fluid barrier. NCBE is predicted to contain at least 10 transmembrane helices. The N- and C- termini are both cytoplasmic, with a large N-terminal domain (Nt-NCBE) and a relatively small C-terminal domain (Ct-NCBE). The Nt-NCBE is likely to be involved in bicarbonate recognition and transport and contains key areas of regulation involving pH sensing and protein-protein interactions. Intrinsic disordered protein regions (IDPRs) are defined as protein regions having no rigid three-dimensional structure under physiological conditions. They are believed to be involved in signaling networks in which specific, low affinity, protein-protein interactions play an important role. We predict that NCBE and other SoLute Carrier 4 (SLC4) family members have a high level of intrinsic disorder in their cytoplasmic regions. To provide biophysical evidence for the IDPRs predicted in Nt-NCBE, we produced pure (>99%), recombinant Nt-NCBE using E. coli as the expression host. The protein was used to perform differential scanning fluorescence spectroscopy (DSF), in order to search for small molecules that would induce secondary or tertiary structure in the IDPRs. We expect this to assist the development of selective pharmaceutical compounds against individual SLC4 family members. We have also determined a low resolution (4 Å) X-ray crystal structure of the N-terminal core domain. The N-terminal cytoplasmic domain (cdb3) of anion exchanger 1 (AE1) shares a similar fold with the N-terminal core domain of NCBE. Crystallization conditions for the full-length N-terminal domain have been sought, but only the core domain yields diffracting crystals.

Differential scanning fluorescence approach using a fluorescent molecular rotor to detect thermostability of proteins in surfactant-containing formulations

A differential scanning fluorimetry (DSF) based high-throughput screening assay with the fluorescent molecular rotor CCVJ (9-(2-carboxy-2-cyanovinyl)julolidine) was developed. CCVJ is mainly sensitive to viscosity and less to polarity in comparison to polarity-sensitive dyes like SYPRO Orange, which was commonly used in DSF measurements. Therefore DSF with CCVJ is a suitable approach for high-throughput screening and stability testing of surfactant-containing protein formulations. Due to the different detection principles of CCVJ and SYPRO Orange, the midpoint of the fluorescence curve of CCVJ, defined as temperature of aggregation (Tagg), was obtained at a higher temperature than the midpoint of the SYPRO Orange fluorescence curve, defined as temperature of hydrophobic exposure (Th). Granulocyte colony stimulating factor (G-CSF) was used as model protein for all measurements. Commonly used surfactants in therapeutic protein formulations (polysorbate 20, polysorbate 80 and poloxamer 188) were investigated by DSF with CCVJ and SYPRO Orange. The fluorescence properties of CCVJ were minimally affected by investigated surfactants at concentrations typically used in pharmaceutical protein formulations. SYPRO Orange however, showed high background fluorescence as it also interacts with hydrophobic groups of surfactants. CCVJ was also capable of detecting thermally induced aggregation in the commercial polysorbate 80-containing product Neupogen(®).

Formulation Development of Therapeutic Monoclonal Antibodies Using High-Throughput Fluorescence and Static Light Scattering Techniques: Role of Conformational and Colloidal Stability

In this work, we describe the application of two different high-throughput screening (HTS) techniques that can be used to determine protein stability during early formulation development. Differential scanning fluorescence (DSF) and differential static light scattering (DSLS) are used to determine the conformational and colloidal stability of therapeutic monoclonal antibodies (mAbs) during thermal denaturation in a high-throughput fashion. DSF utilizes SYPRO Orange, a polarity-sensitive extrinsic fluorescent probe, to monitor protein unfolding. We found that melting temperatures determined by DSF have a linear correlation with melting temperatures of the first domain unfolding determined by differential scanning calorimetry, establishing DSF as a reliable method for measuring thermal stability. The DSLS method employs static light scattering to evaluate protein stability during thermal denaturation in a 384-well format. Overall comparison between mAb aggregation under typical accelerated stress conditions (40°C) and the thermal stability obtained by DSF and DSLS is also presented. Both of these HTS methods are cost effective with high-throughput capability and can be implemented in any laboratory. Combined with other emerging HTS techniques, DSF and DSLS could be powerful tools for mAb formulation optimization.

The Combined Use of the Thermofluor Assay and ThermoQ Analytical Software for the Determination of Protein Stability and Buffer Optimization as an Aid in Protein Crystallization

The Thermofluor assay, also referred to as a thermal shift assay or differential scanning fluorescence (DSF), is a fast and simple method that is based upon high-throughput measurements of protein stability. The Thermofluor method can be performed on nearly all qPCR machines, meaning no special instrumentation is necessary, and can be used to validate the quality of protein preparations, screen for ligands or cofactors, and discover buffers and additives that maximize protein stability. This unit describes how to set up a Thermofluor method on several common models of qPCR instruments, how to prepare the samples for the assay, and how to run and analyze the resulting data. The unit also describes a 96-well screen to determine optimal buffer conditions for protein stability, which may assist in protein crystallization, and details the use of custom software, developed specifically for the analysis of data from Thermofluor screens.

Differential Scanning Fluorimetry Measurement of Protein Stability Changes upon Binding to Glycosaminoglycans: A Screening Test for Binding Specificity

The interaction between glycosaminoglycans (GAGs) and proteins is important for the regulation of protein transport and activity. Here we present a novel method for the measurement of protein-GAG interactions suitable for high-throughput screening, able to discriminate between the interactions of a protein with GAGs of different structures. Binding of proteins to the GAG heparin, a proxy for sulfated regions of extracellular heparan sulfate, was found to enhance the stability of three test proteins, fibroblast growth factors (FGFs)-1, -2, and -18. Chemically modified heparins and heparin oligosaccharides of different lengths stabilized the three FGFs to different extents, depending on the pattern of sugar binding specificity. The method is based on a differential scanning fluorescence approach. It uses a Sypro Orange dye, which binds to exposed core residues of a denatured protein and results in an increased fluorescence signal. It is convenient, requiring low micromolar amounts of protein and ligand compared to other interaction assays, employing only a real-time polymerase chain reaction (PCR) instrument.