Fluorescent Thermal Shift Assay Research Articles

Fluorescence Thermal Shift Assay based High Through-put Screening in Small Molecule Drug Discovery: A Brief Overview

Background: The Early drug discovery process was majorly phenotypic, lacking target specificity, and mechanism of action causing short and/or long-term toxicological impacts on the patients leading to several drug withdrawals from the market. Biochemical procedures and methods used in drug discovery and development make it lengthy in terms of time requiring approx. 15 years with the investment of billions of dollars for a new drug molecule. Technological advancement leading to the identification of protein structures through crystallography paved a new path toward targeted drug discovery. Objective: This paper provides a brief overview of the use of the fluorescence thermal shift assay (FTSA) as a high throughput (HTS) screening technique in small molecule drug discovery. Methods: The article discusses the principles of thermal denaturation techniques and how they are applied in drug discovery. It highlights the advantages of FTSA over other biophysical methods, including its label-free, cost-effective, and high HTS nature. The review also presents a comparative analysis of FTSA with techniques such as isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), X-ray crystallography(X-RD), circular dichroism (CD), and differential scanning calorimetry (DSC). Results: The FTSA technique is described as a powerful tool that addresses the limitations of larger protein requirements, specific assay development, and low HTS associated with other thermal denaturation methods. Unlike biochemical and cellular assays that measure secondary effects, FTSA directly estimates the binding of small molecules to target proteins. Conclusion: The review concludes that FTSA is the only biophysical method that satisfies the three key prerequisites of being label-free, cost-effective, and suitable for high HTS screening in small molecule drug discovery.

Screening, Synthesis and Biochemical Characterization of SARS-CoV-2 Protease Inhibitors.

The severe acute respiratory syndrome-causing coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) and main protease (Mpro) play an important role in viral replication events and are important targets for anti-coronavirus drug discovery. In search of these protease inhibitors, we screened a library of 1300 compounds using a fluorescence thermal shift assay (FTSA) and identified 53 hits that thermally stabilized or destabilized PLpro. The hit compounds structurally belonged to two classes of small molecules: thiazole derivatives and symmetrical disulfide compounds. Compound dissociation constants (Kd) were determined using an enzymatic inhibition method. Seven aromatic disulfide compounds were identified as efficient PLpro inhibitors with Kd values in the micromolar range. Two disulfides displayed six-fold higher potency for PLpro (Kd = 0.5 µM) than for Mpro. The disulfide derivatives bound covalently to both proteases, as confirmed through mass spectrometry. The identified compounds can serve as lead compounds for further chemical optimization toward anti-COVID-19 drugs.

PLBD: protein-ligand binding database ofthermodynamic andkinetic intrinsic parameters.

We introduce a protein-ligand binding database (PLBD) that presents thermodynamic and kinetic data of reversible protein interactions with small molecule compounds. The manually curated binding data are linked to protein-ligand crystal structures, enabling structure-thermodynamics correlations to be determined. The database contains over 5500 binding datasets of 556 sulfonamide compound interactions with the 12 catalytically active human carbonic anhydrase isozymes defined by fluorescent thermal shift assay, isothermal titration calorimetry, inhibition of enzymatic activity and surface plasmon resonance. In the PLBD, the intrinsic thermodynamic parameters of interactions are provided, which account for the binding-linked protonation reactions. In addition to the protein-ligand binding affinities, the database provides calorimetrically measured binding enthalpies, providing additional mechanistic understanding. The PLBD can be applied to investigations of protein-ligand recognition and could be integrated into small molecule drug design. Database URL https://plbd.org/.

Designing, synthesis and evaluation of derived analogues of selected small molecule non-peptidic inhibitors against serotype BoNT/ F

Botulinum neurotoxins are lethal Biowarfare categorized in group A of selected agents, by CDC USA. The unavailability of counter-measures against these neurotoxins has been a matter of extensive research. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. We have reported 8-HQ compounds NSC1014 and NSC1011 as potential small molecule inhibitors against BoNT/F. In the present study, analogues of NSC84087 and NSC1014 were designed, synthesized and studied for their inhibitory role against BoNT/F intoxication through in silico study, in vitro and in-vivo assays. ∼25 in-house synthesized small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay and then further assessed through endopeptidase assay. The binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in-vivo efficacy of these compounds was evaluated in mice model. Analogues C87.9, C87.10 and C87.12 of compound NSC84087 and C14.10, C14.11 and C14.13 of NSC1014 showed promising results through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed KD values in sub-micromolar range signifying high affinity interaction. The IC50 of C14.10 was found to be the lowest of 3.016 ± 0.798 μM as determined through endopeptidase assay. Finally, efficacy of selected molecules was evaluated in mice, C14.10 and C14.13 protected 40% animals against 4X LD50 and extended survival time up to 200% at 10X LD50. The present study thus proposes the emergence of NSC84087 and NSC1014 analogues as lead compound against BoNT/F.

Validation of the solution structure of dimerization domain of PRC1.

Cell-cycle dependent proteins are indispensible for the accurate division of cells, a group of proteins called Microtubule-associated proteins (MAPs) are important to cell division as it bind microtubules and participate with other co-factors to form the spindle midbody, which works as the workhorse of cell-division. PRC1 is a distinguishing member of MAPs, as it is a human MAP and works as the key in mediating daughter cell segregation in ana-phase and telo-phase. The physiological significance of PRC1 calls for a high resolution three-dimensional structure. The crystal structure of PRC1 was published but has low resolution (>3 Å) and incomplete sidechains, placing hurdles to understanding the structure-function relationships of PRC1, therefore, we determined the high-resolution solution structure of PRC1’s dimerization domain using NMR spectroscopy. Significant differences between the crystal structure and the solution structure can be observed, the main differences center around the N terminus and the end of the alpha-Helix H2. Furthermore, detailed structure analyses revealed that the hydrophobic core packing of the solution and crystal structures are also different. To validate the solution structure, we used Hydrogen-deuterium exchange experiments that address the structural discrepancies between the crystal and solution structure; we also generated mutants that are key to the differences in the crystal and solution structures, measuring its structural or thermal stability by NMR spectroscopy and Fluorescence Thermal Shift Assays. These results suggest that N terminal residues are key to the integrity of the whole protein, and the solution structure of the dimerization domain better reflects the conformation PRC1 adopted in solution conditions.

Drug discovery of small molecules targeting the higher-order hTERT promoter G-quadruplex.

DNA G-quadruplexes (G4s) are now widely accepted as viable targets in the pursuit of anticancer therapeutics. To date, few small molecules have been identified that exhibit selectivity for G4s over alternative forms of DNA, such as the ubiquitous duplex. We posit that the lack of current ligand specificity arises for multiple reasons: G4 atomic models are often small, monomeric, single quadruplex structures with few or no druggable pockets; targeting G-tetrad faces frequently results in the enrichment of extended electron-deficient polyaromatic end-pasting scaffolds; and virtual drug discovery efforts often under-sample chemical search space. We show that by addressing these issues we can enrich for non-standard molecular templates that exhibit high selectivity towards G4s over other forms of DNA. We performed an extensive virtual screen against the higher-order hTERT core promoter G4 that we have previously characterized, targeting 12 of its unique loop and groove pockets using libraries containing 40 million drug-like compounds for each screen. Using our drug discovery funnel approach, which utilizes high-throughput fluorescence thermal shift assay (FTSA) screens, microscale thermophoresis (MST), and orthogonal biophysical methods, we have identified multiple unique G4 binding scaffolds. We subsequently used two rounds of catalogue-based SAR to increase the affinity of a disubstituted 2-aminoethyl-quinazoline that stabilizes the higher-order hTERT G-quadruplex by binding across its G4 junctional sites. We show selectivity of its binding affinity towards hTERT is virtually unaffected in the presence of near-physiological levels of duplex DNA, and that this molecule downregulates hTERT transcription in breast cancer cells.

Identifying New Ligands for JNK3 by Fluorescence Thermal Shift Assays and Native Mass Spectrometry.

The c-Jun N-terminal kinases (JNKs) are evolutionary highly conserved serine/threonine kinases. Numerous findings suggest that JNK3 is involved in the pathogenesis of neurodegenerative diseases, so the inhibition of JNK3 may be a potential therapeutic intervention. The identification of novel compounds with promising pharmacological properties still represents a challenge. Fluorescence thermal shift screening of a chemically diversified lead-like scaffold library of 2024 pure compounds led to the initial identification of seven JNK3 binding hits, which were classified into four scaffold groups according to their chemical structures. Native mass spectrometry validated the interaction of 4 out of the 7 hits with JNK3. Binding geometries and interactions of the top 2 hits were evaluated by docking into a JNK3 crystal structure. Hit 5 had a Kd of 21 μM with JNK3 suggested scaffold 5-(phenylamino)-1H-1,2,3-triazole-4-carboxamide as a novel and selective JNK3 binder.

Beta and Gamma Amino Acid-Substituted Benzenesulfonamides as Inhibitors of Human Carbonic Anhydrases.

A series of novel benzenesulfonamide derivatives were synthesized bearing para-N β,γ-amino acid or para-N β-amino acid and thiazole moieties and their binding to the human carbonic anhydrase (CA) isozymes determined. These enzymes are involved in various illnesses, such as glaucoma, altitude sickness, epilepsy, obesity, and even cancer. There are numerous compounds that are inhibitors of CA and used as pharmaceuticals. However, most of them bind to most CA isozymes with little selectivity. The design of high affinity and selectivity towards one CA isozyme remains a significant challenge. The beta and gamma amino acid-substituted compound affinities were determined by the fluorescent thermal shift assay and isothermal titration calorimetry for all 12 catalytically active human carbonic anhydrase isozymes, showing the full affinity and selectivity profile. The structures of several compounds were determined by X-ray crystallography, and the binding mode in the active site of CA enzyme was shown.

N-Terminus-Mediated Solution Structure of Dimerization Domain of PRC1.

Simple SummaryThe solution structure of the N-terminal domain of Protein Regulator of Cytokinesis 1 (PRC1) was determined, and compared with the previously published crystal structure, significant differences were found. Extensive analyses were carried out to find the true reason for the differences between the solution and crystal structures, we discovered that this might be related to the conformation of residue M1, which is buried in the protein core of the solution structure, while situated outside of the hydrophobic core in the crystal structure. In this study, we have carried out a series of examinations using various methods and confirmed that the N terminal conformation is the key point in better describing the structure of PRC1 dimerization domain under solution conditions.Microtubule-associated proteins (MAPs) are essential for the accurate division of a cell into two daughter cells. These proteins target specific microtubules to be incorporated into the spindle midzone, which comprises a special array of microtubules that initiate cytokinesis during anaphase. A representative member of the MAPs is Protein Regulator of Cytokinesis 1 (PRC1), which self-multimerizes to cross-link microtubules, the malfunction of which might result in cancerous cells. The importance of PRC1 multimerization makes it a popular target for structural studies. The available crystal structure of PRC1 has low resolution (>3 Å) and accuracy, limiting a better understanding of the structure-related functions of PRC1. Therefore, we used NMR spectroscopy to better determine the structure of the dimerization domain of PRC1. The NMR structure shows that the PRC1 N terminus is crucial to the overall structure integrity, but the crystal structure bespeaks otherwise. We systematically addressed the role of the N terminus by generating a series of mutants in which N-terminal residues methionine (Met1) and arginine (Arg2) were either deleted, extended or substituted with other rationally selected amino acids. Each mutant was subsequently analyzed by NMR spectroscopy or fluorescence thermal shift assays for its structural or thermal stability; we found that N-terminal perturbations indeed affected the overall protein structure and that the solution structure better reflects the conformation of PRC1 under solution conditions. These results reveal that the structure of PRC1 is governed by its N terminus through hydrophobic interactions with other core residues, such hitherto unidentified N-terminal conformations might shed light on the structure–function relationships of PRC1 or other proteins. Therefore, our study is of major importance in terms of identifying a novel structural feature and can further the progress of protein folding and protein engineering.

Design and Synthesis of Hydrazone‐Bearing Benzenesulfonamides as Carbonic Anhydrase VB Inhibitors

AbstractThe α‐amino acid derivatives are constituents of many bioactive compounds and display a wide variety of biological activities. We have synthesized a series of new benzenesulfonamide derivatives bearing α‐amino acid moiety at para‐position and evaluated their binding affinity to human carbonic anhydrase isozymes by fluorescent thermal shift assay. The dichloro‐ and monobromo‐substitutions on the benzenesulfonamide ring have been introduced to determine the halogenation effect on the binding affinity. Chloro substituents at 3,5‐positions of benzenesulfonamide derivatives increased the affinity for all carbonic anhydrases as compared to non‐chlorinated compounds. The hydrazone‐bearing 3,5‐dichlorobenzenesulfonamides 9 a, 9 d, and 12 exhibited a low nanomolar affinity for CA VB (Kd in the range of 6.6–8.1 nM), an isozyme implicated in diseases of the central nervous system and obesity.

Nucleosome Assembly and Disassembly in vitro Are Governed by Chemical Kinetic Principles.

As the elementary unit of eukaryotic chromatin, nucleosomes in vivo are highly dynamic in many biological processes, such as DNA replication, repair, recombination, or transcription, to allow the necessary factors to gain access to their substrate. The dynamic mechanism of nucleosome assembly and disassembly has not been well described thus far. We proposed a chemical kinetic model of nucleosome assembly and disassembly in vitro. In the model, the efficiency of nucleosome assembly was positively correlated with the total concentration of histone octamer, reaction rate constant and reaction time. All the corollaries of the model were well verified for the Widom 601 sequence and the six artificially synthesized DNA sequences, named CS1–CS6, by using the salt dialysis method in vitro. The reaction rate constant in the model may be used as a new parameter to evaluate the nucleosome reconstitution ability with DNAs. Nucleosome disassembly experiments for the Widom 601 sequence detected by Förster resonance energy transfer (FRET) and fluorescence thermal shift (FTS) assays demonstrated that nucleosome disassembly is the inverse process of assembly and can be described as three distinct stages: opening phase of the (H2A–H2B) dimer/(H3–H4)2 tetramer interface, release phase of the H2A–H2B dimers from (H3–H4)2 tetramer/DNA and removal phase of the (H3–H4)2 tetramer from DNA. Our kinetic model of nucleosome assembly and disassembly allows to confirm that nucleosome assembly and disassembly in vitro are governed by chemical kinetic principles.

Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases.

Zinc-containing metalloenzyme carbonic anhydrase (CA) binds primary sulfonamides with extremely high, up to picomolar, affinity by forming a coordination bond between the negatively charged amino group and the zinc ion and making hydrogen bonds and hydrophobic contacts with other parts of the inhibitor molecule. However, N-methyl-substituted, secondary or tertiary sulfonamides bind CA with much lower affinity. In search for an explanation for this diminished affinity, a series of secondary sulfonamides were synthesized and, together with analogous primary sulfonamides, the affinities for 12 recombinant catalytically active human CA isoforms were determined by the fluorescent thermal shift assay, stopped-flow assay of the inhibition of enzymatic activity and isothermal titration calorimetry. The binding profile of secondary sulfonamides as a function of pH showed the same U-shape dependence seen for primary sulfonamides. This dependence demonstrated that there were protein binding-linked protonation reactions that should be dissected for the estimation of the intrinsic binding constants to perform structure-thermodynamics analysis. X-ray crystallographic structures of secondary sulfonamides and computational modeling dissected the atomic contributions to the binding energetics. Secondary sulfonamides bind to carbonic anhydrases via coordination bond between the negatively charged nitrogen of alkylated amino group and Zn(II) in the active site of CA. The binding reaction is linked to deprotonation of the amino group and protonation of the Zn(II)-bound hydroxide. To perform the structure-thermodynamics analysis, contributions of these linked reactions must be subtracted to determine the intrinsic energetics. In this aspect, the secondary sulfonamides are similar to primary sulfonamides as CA inhibitors.

Thiazide and other Cl-benzenesulfonamide-bearing clinical drug affinities for human carbonic anhydrases.

Twelve carbonic anhydrase (CA) isoforms catalyze carbon dioxide hydration to bicarbonate and acid protons and are responsible for many biological functions in human body. Despite their vital functions, they are also responsible for, or implicated in, numerous ailments and diseases such as glaucoma, high altitude sickness, and cancer. Because CA isoforms are highly homologous, clinical drugs designed to inhibit enzymatic activity of a particular isoform, can also bind to others with similar affinity causing toxic side effects. In this study, the affinities of twelve CA isoforms have been determined for nineteen clinically used drugs used to treat hypertension related diseases, i.e. thiazides, indapamide, and metolazone. Their affinities were determined using a fluorescent thermal shift assay. Stopped flow assay and isothermal titration calorimetry were also employed on a subset of compounds and proteins to confirm inhibition of CA enzymatic activity and verify the quantitative agreement between different assays. The findings of this study showed that pharmaceuticals could bind to human CA isoforms with variable affinities and inhibit their catalytic activity, even though the drug was intended to interact with a different (non-CA) protein target. Relatively minor structural changes of the compounds may cause significant changes in affinity and selectivity for a particular CA isoform.

Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein–ligand binding and protein stability

A standard operating procedure for a fluorescence-based thermal shift assay (FTSA) is provided describing its typical applications, advantages and limitations. FTSA is a simple, robust, universal and quick assay to determine protein-ligand binding affinities and protein stabilities in the presence of various excipients and solution conditions. Therefore, the assay is very useful for the straightforward characterization of new recombinantly produced proteins. The assay has a wide dynamic range enabling simultaneous determination of affinities in the milimolar to picomolar range. The assay could be used for essentially any protein that is sufficiently soluble and stable in the studied aqueous solution. Here we provide examples and typical experimental protocols for both affinity and stability determinations.

POT1 stability and binding measured by fluorescence thermal shift assays.

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 -DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

Experimental Approaches to Identify Selective Picomolar Inhibitors for Carbonic Anhydrase IX.

Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible hydration of CO2, thereby emerging as essential enzymes for many vital functions. Among 12 catalytically active CA isoforms in humans, CA IX has become a relevant therapeutic target because of its role in cancer progression. Only two CA IX inhibitors have entered clinical trials, mostly due to low affinity and selectivity properties. The current review presents the design, development, and identification of the selective nano- to picomolar CA IX inhibitors VD11-4-2, VR16-09, and VD12-09. Compounds were selected from our database, composed of over 400 benzensulfonamides, synthesized at our laboratory, and tested for their binding to 12 human CAs. Here we discuss the CA CO2 hydratase activity/inhibition assay and several biophysical techniques, such as fluorescent thermal shift assay and isothermal titration calorimetry, highlighting their contribution to the analysis of compound affinity and structure- activity relationships. To obtain sufficient amounts of recombinant CAs for inhibitor screening, several gene cloning and protein purification strategies are presented, including site-directed CA mutants, heterologous CAs from Xenopus oocytes, and native endogenous CAs. The cancer cell-based methods, such as clonogenicity, extracellular acidification, and mass spectrometric gas-analysis are reviewed, confirming nanomolar activities of lead inhibitors in intact cancer cells. Novel CA IX inhibitors are promising derivatives for in vivo explorations. Furthermore, the simultaneous targeting of several proteins involved in proton flux upon tumor acidosis and the disruption of transport metabolons might improve cancer management.

Targeted 8-hydroxyquinoline fragment based small molecule drug discovery against neglected botulinum neurotoxin type F

ObjectivesBotulinum neurotoxins are highly potent biological warfare agents. The unavailability of countermeasures against these neurotoxins has been a matter of extensive research. However, no clinical therapeutics has come to existence till date. The 8-hydroxyquinoline (8-HQ) scaffold is established privileged compound and its potential as drug candidate against BoNTs is recently being explored. MethodsIn present work, three course studies were performed involving in silico, in vitro and in vivo cascade to screen 8-HQ small molecule inhibitors against BoNT/F intoxication. ~800 molecules obtained from open repositories were screened in silico and commercially obtained twenty-four 8-HQ derived small molecule inhibitors were evaluated against rBoNT/F light chain through fluorescence thermal shift (FTS) assay. Selected compounds were further evaluated through endopeptidase assay. Further binding affinity analysis was done through surface plasmon resonance (SPR) based Proteon™ XPR 36 system. Finally, the in vivo efficacy of these compounds was evaluated in mice model. ResultsThree compounds NSC1011, NSC1014 and NSC84094 were found to be highly inhibitory after screening of 8-HQ compounds through FTS assay and endopeptidase assay. SPR based protein-small molecule interaction studies showed highest affinity binding of NSC1014 (KD: 5.58E-06) with BoNT/F-LC. NSC1011, NSC1014, and NSC84094 displayed IC50 of 30.47 ± 6.24, 14.91 ± 2.49 and 17.39 ± 2.74 μM, respectively, in endopeptidase assay. NSC1011 and NSC1014 displayed marked extension of survival time in mice model. ConclusionNSC1011 and NSC1014 have emerged as promising drug candidate against BoNT/F intoxication displaying higher potential than previously reported compounds.

Engineered Carbonic Anhydrase VI-Mimic Enzyme Switched the Structure and Affinities of Inhibitors

Secretory human carbonic anhydrase VI (CA VI) has emerged as a potential drug target due to its role in pathological states, such as excess acidity-caused dental caries and injuries of gastric epithelium. Currently, there are no available CA VI-selective inhibitors or crystallographic structures of inhibitors bound to CA VI. The present study focuses on the site-directed CA II mutant mimicking the active site of CA VI for inhibitor screening. The interactions between CA VI-mimic and a series of benzenesulfonamides were evaluated by fluorescent thermal shift assay, stopped-flow CO2 hydration assay, isothermal titration calorimetry, and X-ray crystallography. Kinetic parameters showed that A65T, N67Q, F130Y, V134Q, L203T mutations did not influence catalytic properties of CA II, but inhibitor affinities resembled CA VI, exhibiting up to 0.16 nM intrinsic affinity for CA VI-mimic. Structurally, binding site of CA VI-mimic was found to be similar to CA VI. The ligand interactions with mutated side chains observed in three crystallographic structures allowed to rationalize observed variation of binding modes and experimental binding affinities to CA VI. This integrative set of kinetic, thermodynamic, and structural data revealed CA VI-mimic as a useful model to design CA VI-specific inhibitors which could be beneficial for novel therapeutic applications.

Pyrrolidinone-bearing methylated and halogenated benzenesulfonamides as inhibitors of carbonic anhydrases.

Two series of benzenesulfonamides bearing methyl groups at ortho/ortho or meta/ortho positions and a pyrrolidinone moiety at para position were synthesized and tested as inhibitors of the twelve catalytically active human carbonic anhydrase (CA) isoforms. Observed binding affinities were determined by fluorescent thermal shift assay and intrinsic binding affinities representing the binding of benzenesulfonamide anion to the Zn(II)-bound water form of CA were calculated. Introduction of dimethyl groups into benzenesulfonamide ring decreased the binding affinity to almost all CA isoforms, but gained in selectivity towards one CA isoform. A chloro group at the meta position of 2,6-dimethylbenzenesulfonamide derivatives did not influence the binding to CA I, but it increased the affinity to all other CAs, especially, CA VII and CA XIII (up to 500 fold). The compounds may be used for further development of CA inhibitors with higher selectivity to particular CA isoforms.

Small Molecule Inhibitor that Stabilizes the Autoinhibited Conformation of the Oncogenic Tyrosine Phosphatase SHP2.

Genetic mutations in the phosphatase PTPN11 (SHP2) are associated with childhood leukemias. These mutations cause hyperactivation of SHP2 due to the disruption of the autoinhibitory conformation. By targeting the activation-associated protein conformational change, we have identified an SHP2 inhibitor ( E)-1-(1-(5-(3-(2,4-dichlorophenyl)acryloyl)-2-ethoxy-4-hydroxybenzyl)-1,2,5,6-tetrahydropyridin-3-yl)-1 H-benzo[ d]imidazol-2(3 H)-one (LY6, 1) using computer-aided drug design database screening combined with cell-based assays. This compound inhibited SHP2 with an IC50 value of 9.8 μM, 7-fold more selective for SHP2 than the highly related SHP1. Fluorescence titration, thermal shift, and microscale thermophoresis quantitative binding assays confirmed its direct binding to SHP2. This compound was further verified to effectively inhibit SHP2-mediated cell signaling and proliferation. Furthermore, mouse and patient leukemia cells with PTPN11 activating mutations were more sensitive to this inhibitor than wild-type cells. This small molecule SHP2 inhibitor has a potential to serve as a lead compound for further optimization studies to develop novel anti-SHP2 therapeutic agents.