From X-ray crystallographic structure to intrinsic thermodynamics of protein-ligand binding using carbonic anhydrase isozymes as a model system.

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery.

Recent advances in computational and experimental protein-ligand affinity determination techniques.

INTRODUCTION: Modern drug discovery revolves around designing ligands that target the chosen biomolecule, typically proteins. For this, the evaluation of affinities of putative ligands is crucial. This has given rise to a multitude of dedicated computational and experimental methods that are constantly being developed and improved. AREAS COVERED: In this review, the authors reassess both the industry mainstays and the newest trends among the methods for protein - small-molecule affinity determination. They discuss both computational affinity predictions and experimental techniques, describing their basic principles, main limitations, and advantages. Together, this serves as initial guide to the currently most popular and cutting-edge ligand-binding assays employed in rational drug design. EXPERT OPINION: The affinity determination methods continue to develop toward miniaturization, high-throughput, and in-cell application. Moreover, the availability of data analysis tools has been constantly increasing. Nevertheless, cross-verification of data using at least two different techniques and careful result interpretation remain of utmost importance.

High-Affinity NIR-Fluorescent Inhibitors for Tumor Imaging via Carbonic Anhydrase IX.

Tumor imaging and delivery of therapeutic agents may be achieved by designing high-affinity and high-selectivity compounds recognizing a tumor cell-expressing biomarker, such as carbonic anhydrase IX (CA IX). The CAIX, overexpressed in many hypoxic solid tumors, helps adjust to the energy requirements of the hypoxic environment, reduces intracellular acidification, and participates in the metastatic invasion of adjacent tissues. Here, we designed a series of sulfonamide compounds bearing CAIX-recognizing, high-affinity, and high-selectivity groups conjugated via a PEG linker to near-infrared (NIR) fluorescent probes used in the clinic for optically guided cancer surgery. We determined compound affinities for CAIX and other 11 catalytically active CA isozymes by the thermal shift assay and showed that the affinity Kd value of CAIX was in the subnanomolar range, hundred to thousand-fold higher than those of other CA isozymes. Similar affinities were also observed for CAIX expressed on the cancer cell surface in live HeLa cell cultures, as determined by the competition assay. The NIR-fluorescent compounds showed excellent properties in visualizing CAIX-positive tumors but not CAIX-negative knockout tumors in a nude mice xenograft model. These compounds would therefore be helpful in optically guided cancer surgery and could potentially be developed for anticancer treatment by radiotherapy.

Comprehensive analysis of resorcinyl-imidazole Hsp90 inhibitor design.

Human Hsp90 chaperones are implicated in various aspects of cancer. Due to this, Hsp90 has been explored as potential target in cancer treatment. Initial attempts to use Hsp90 inhibitors in drug trials failed due to toxicity and inefficacy. The next generation of drugs were less toxic but still insufficiently effective in a clinical setting. Recently, a lot of effort is being put into understanding the consequences of Hsp90 isoform selective inhibition, expecting that this might hold the key in targeting Hsp90 for disease treatment. Here we investigate a series of compounds containing the aryl-resorcinol scaffold with a 5-membered ring as a promising class of new human Hsp90 inhibitors, reaching nanomolar affinity. We compare how the replacement of 5-membered ring, from thiadiazole to imidazole, as well as a variety of their substituents, influences the potency of these inhibitors for Hsp90 alpha and beta isoforms. To further elucidate the dissimilarity in ligand selectivity between the isoforms, a mutant protein was constructed and tested against the ligand library. In addition, we performed a series of molecular dynamics (MD) and docking simulations to further explain our experimental findings as well as evaluated key compounds in cell assays. Our results deepen the understanding of Hsp90 isoform ligand selectivity and serve as an informative base for further Hsp90 inhibitor optimization.

Aerobic vaginitis is associated with carbonic anhydrase IX in cervical intraepithelial neoplasia.

The aim of this study was to analyze the association between vaginal microbiota, carbonic anhydrase IX (CAIX) and histological findings of cervical intraepithelial neoplasia (CIN). The study included 132 females, among them 66 were diagnosed with high-grade intraepithelial lesion (CIN2, CIN3, and cancer), 14 with low-grade disease, and 52 assigned to the control group. An interview focused on the behavior risk factors, together with vaginal fluid pH measurement, wet mount microscopy, detection of Chlamydia trachomatis, and Trichomonas vaginalis were performed. After colposcopy, high-grade abnormalities were detected via direct biopsies and treated with conization procedure. Conuses were immuno-stained with CAIX antibody. The histological findings were CIN1 (n = 14), and CIN2+ (included CIN2 (n = 10), CIN3 (n = 49), and cancer (n = 7; squamous cell carcinomas)). Prevalence of bacterial vaginosis (BV) was similar between the groups. Moderate or severe aerobic vaginitis (msAV) was diagnosed more often among CIN2+ (53.0%) than CIN1 (21.4%). Moderate or strong immunostaining of CAIX (msCAIX) was not detected among CIN1 cases. Thus, msAV was prevalent in CAIX non-stained group (p = 0.049) among CIN2 patients. Co-location of msAV and msCAIX was found in CIN3. Regression model revealed that msAV associated with high-grade cervical intraepithelial neoplasia independently from smoking and the number of partners.

Inhibitor binding to metal-substituted metalloenzyme: Sulfonamide affinity for carbonic anhydrase IX.

Transition metal ions are structural and catalytic cofactors of many proteins including human carbonic anhydrase (CA), a Zn-dependent hydrolase. Sulfonamide inhibitors of CA recognize and form a coordination bond with the Zn ion located in the active site of the enzyme. The Zn ion may be removed or substituted with other metal ions. Such CA protein retains the structure and could serve as a tool to study metal ion role in the recognition and binding affinity of inhibitor molecules. We measured the affinities of selected divalent transition metal ions, including Mn, Fe, Co, Ni, Cu, Cd, Hg, and Zn to metal-free CA isozymes CA I, CA II, and CAIX by fluorescence-based thermal shift assay, prepared metal-substituted CAs, and determined binding of diverse sulfonamide compounds. Sulfonamide inhibitor binding to metal substituted CA followed a U-shape pH dependence. The binding was dissected to contributing binding-linked reactions and the intrinsic binding reaction affinity was calculated. This value is independent of pH and protonation reactions that occur simultaneously upon binding native CA and as demonstrated here, to metal substituted CA. Sulfonamide inhibitor binding to cancer-associated isozyme CAIX diminished in the order: Zn > Co > Hg > Cu > Cd > Mn > Ni. Energetic contribution of the inhibitor-metal coordination bond was determined for all above metals. The understanding of the principles of metal influence on ligand affinity and selectivity should help design new drugs targeting metalloenzymes.

Intrinsic Solubility of Ionizable Compounds from pKa Shift.

Aqueous solubility of pharmaceutical substances plays an important role in small molecule drug discovery and development, with ionizable groups often employed to enhance solubility. Drug candidate compounds often contain ionizable groups to increase their solubility. Recognizing that the electrostatically charged form of the compound is much more soluble than the uncharged form, this work proposes a model to explore the relationship between the pKa shift of the ionizable group and dissolution equilibria. The model considers three forms of a compound: dissolved-charged, dissolved-uncharged, and aggregated-uncharged. It analyzes two linked equilibria: the protonation of the ionizable group and the dissolution-aggregation of the uncharged form, with the observed pKa shift depending on the total concentration of the compound. The active concentration of the aggregates determines this shift. The model was explored through the determination of the pKa shift and intrinsic solubility of specific compounds, such as ICPD47, a high-affinity inhibitor of the Hsp90 chaperone protein and anticancer target, as well as benzoic acid and benzydamine. The model holds the potential for a more nuanced understanding of intrinsic solubility and may lead to advancements in drug discovery and development.

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 of thermodynamic and kinetic 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/.

Special Issue "Advances in Monitoring Metabolic Activities of Microorganisms by Calorimetry".

In recent decades, the calorimetric monitoring of microbial metabolism, i [...].

Inhibitory effects of fluorinated benzenesulfonamides on insulin fibrillation.

Amyloid fibrils are protein aggregates formed by protein assembly through cross ß structures. Inhibition of amyloid fibril formation may contribute to therapy against amyloid-related disorders like Parkinson's, Alzheimer's, and type 2 diabetes. Here we report that several fluorinated sulfonamide compounds, previously shown to inhibit human carbonic anhydrase, also inhibit the fibrillation of different proteins. Using a range of spectroscopic, microscopic and chromatographic techniques, we found that the two fluorinated sulfonamide compounds completely inhibit insulin fibrillation over a period of 16 h and moderately suppress α-synuclein and Aß fibrillation. In addition, these compounds decreased cell toxicity of insulin incubated under fibrillation-inducing conditions. We ascribe these effects to their ability to maintain insulin in the native monomeric state. Molecular dynamic simulations suggest that these compounds inhibit insulin self-association by interacting with residues at the dimer interface. This highlights the general anti-aggregative properties of aromatic sulfonamides and suggests that sulfonamide compounds which inhibit carbonic anhydrase activity may have potential as therapeutic agents against amyloid-related disorders.

Picomolar fluorescent probes for compound affinity determination to carbonic anhydrase IX expressed in live cancer cells.

Numerous human cancers, especially hypoxic solid tumors, express carbonic anhydrase IX (CAIX), a transmembrane protein with its catalytic domain located in the extracellular space. CAIX acidifies the tumor microenvironment, promotes metastases and invasiveness, and is therefore considered a promising anticancer target. We have designed a series of high affinity and high selectivity fluorescein-labeled compounds targeting CAIX to visualize and quantify CAIX expression in cancer cells. The competitive binding model enabled the determination of common CA inhibitors' dissociation constants for CAIX expressed in exponentially growing cancer cells. All tested sulfonamide compounds bound the proliferating cells with similar affinity as to recombinantly purified CAIX. The probes are applicable for the design of selective drug-like compounds for CAIX and the competition strategy could be applied to other drug targets.

Factors, associated with elevated concentration of soluble carbonic anhydrase IX in plasma of women with cervical dysplasia.

Precancerous lesions of human cervix uteri have a tendency for regression or progression. In cervical intraepithelial neoplasia grade 2 (CINII) case there is an uncertainty if a lesion will progress or regress. The carbonic anhydrase IX (CAIX) enzyme is overexpressed in cervical cancer which is more sensitive to radiotherapy. CAIX is associated with poor prognosis in solid hypoxic tumors. The aim of this study was to determine factors related to elevated soluble CAIX (s-CAIX) in high-grade intraepithelial lesion (HSIL) cases. METHODS: Patients diagnosed with HSIL (N = 77) were included into the research group whereas without HSIL (N = 72)-the control group. Concentration of the soluble CAIX (s-CAIX) in plasma was determined by the DIANA ligand-antibody-based method. C. trachomatis was detected from cervical samples by PCR. Primary outcomes were risk factors elevating s-CAIX level in HSIL group. Non-parametric statistical analysis methods were used to calculate correlations. RESULTS: The s-CAIX level in patients with HSIL was elevated among older participants (rs = 0.27, p = 0.04) and with C. trachomatis infection (p = 0.028). Among heavy smokers with HSIL, the concentration of s-CAIX was higher in older women (rs = 0.52, p = 0.005), but was not related to the age of heavy smokers' controls (τ = 0.18 p = 0.40). CONCLUSION: The concentration of s-CAIX was higher among older, heavy smoking and diagnosed with C. trachomatis patients. All these factors increased the risk for HSIL progression.

3-Chloro-5-Substituted-1,2,4-Thiadiazoles (TDZs) as Selective and Efficient Protein Thiol Modifiers.

The study of cysteine modifications has gained much attention in recent years. This includes detailed investigations in the field of redox biology with focus on numerous redox derivatives like nitrosothiols, sulfenic acids, sulfinic acids and sulfonic acids resulting from increasing oxidation, S-lipidation, and perthiols. For these studies selective and rapid blocking of free protein thiols is required to prevent disulfide rearrangement. In our attempt to find new inhibitors of human histone deacetylase 8 (HDAC8) we discovered 5-sulfonyl and 5-sulfinyl substituted 1,2,4-thiadiazoles (TDZ), which surprisingly show an outstanding reactivity against thiols in aqueous solution. Encouraged by these observations we investigated the mechanism of action in detail and show that these compounds react more specifically and faster than commonly used N-ethyl maleimide, making them superior alternatives for efficient blocking of free thiols in proteins. We show that 5-sulfonyl-TDZ can be readily applied in commonly used biotin switch assays. Using the example of human HDAC8, we demonstrate that cysteine modification by a 5-sulfonyl-TDZ is easily measurable using quantitative HPLC/ESI-QTOF-MS/MS, and allows for the simultaneous measurement of the modification kinetics of seven solvent-accessible cysteines in HDAC8.

Intrinsic affinity of protein - ligand binding by differential scanning calorimetry.

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein-ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.

Synthesis and HDAC inhibitory activity of pyrimidine-based hydroxamic acids.

Histone deacetylases (HDACs) play an essential role in the transcriptional regulation of cells through the deacetylation of nuclear histone and non-histone proteins and are promising therapeutic targets for the treatment of various diseases. Here, the synthesis of new compounds in which a hydroxamic acid residue is attached to differently substituted pyrimidine rings via a methylene group bridge of varying length as potential HDAC inhibitors is described. The target compounds were obtained by alkylation of 2-(alkylthio)pyrimidin-4(3H)-ones with ethyl 2-bromoethanoate, ethyl 4-bromobutanoate, or methyl 6-bromohexanoate followed by aminolysis of the obtained esters with hydroxylamine. Oxidation of the 2-methylthio group to the methylsulfonyl group and following treatment with amines resulted in the formation of the corresponding 2-amino-substituted derivatives, the ester group of which reacted with hydroxylamine to give the corresponding hydroxamic acids. The synthesized hydroxamic acids were tested as inhibitors of the HDAC4 and HDAC8 isoforms. Among the synthesized pyrimidine-based hydroxamic acids N-hydroxy-6-[6-methyl-2-(methylthio)-5-propylpyrimidin-4-yloxy]hexanamide was found to be the most potent inhibitor of both the HDAC4 and HDAC8 isoforms, with an IC50 of 16.6 µM and 1.2 µM, respectively.

Denaturant- or ligand-induced changes in protein volume by pressure shift assay.

A complete thermodynamic description of protein-ligand binding includes parameters related to pressure and temperature. The changes in the protein volume and compressibility upon binding a ligand are pressure-related parameters that are often neglected due to the lack of routine methods for their determination. Fluorescent pressure shift assay (FPSA) is based on pressure-induced protein unfolding and its stabilization by a ligand and offers a universal approach to determine protein-ligand binding volumes. Extremely high pressures are required to unfold most proteins and protein-ligand complexes. Thus, guanidinium hydrochloride (GdmHCl) is used as a protein-destabilizing agent. We determined that GdmHCl unfolds carbonic anhydrase isoforms in a different pathway, but the destabilization effect is linear in a particular concentration range. We developed a concept for the FPSA experiment, where both - the ligand and GdmHCl - concentrations are varied. This approach enabled us to determine protein-ligand binding volumes that otherwise would be impossible due to the equipment-unreachable pressures of protein unfolding.

Thermott: A comprehensive online tool for protein-ligand binding constant determination.

The thermal shift assay is one of the most universal techniques to determine protein-ligand affinities ranging from millimolar to picomolar levels in a single ligand dosing experiment. However, the complexity of thermodynamic data analysis leads to an underuse of this technique. We have developed a user-friendly, open-source, free online analysis software to study any protein-ligand interaction thermal shift data and yield a comprehensive thermodynamic characterization of the binding reaction.

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

A series of novel benzenesulfonamide derivatives were synthesized bearing para-N ß,γ-amino acid or para-N ß-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.

Inhibition of Carbonic Anhydrase IX Suppresses Breast Cancer Cell Motility at the Single-Cell Level.

Protein Carbonic Anhydrase IX (CA IX), which is expressed in various hypoxic solid tumors in order to maintain proper pH, is also related to cancer cell adhesion, invasion, and metastasis processes. Here, we investigated whether CA IX inhibition by a highly CA IX selective agent benzenesulfonamide VD11-4-2 triggers changes in individual cell motility. We seeded breast cancer cells on an extracellular matrix-coated glass-bottomed dish and in a microfluidic device with a gradient flow of epidermal growth factor (EGF), tracked individual cell movement, calculated their migration speeds, and/or followed movement direction. Our results showed that the inhibitor VD11-4-2 decreased the speed of CA IX positive breast cancer cells by 20-26% while not affecting non-cancerous cell migration. The inhibitor suppressed the cell migration velocity increment and hindered cells from reaching their maximum speed. VD11-4-2 also reduced CA IX, expressing cell movement towards the growth factor as a chemoattractant. Such a single cell-based migration assay enabled the comprehensive investigation of the cell motility and revealed that VD11-4-2 shows the ability to suppress breast cancer cell migration at a lower concentration than previously tested CA IX inhibitors.