RESUMO
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.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Inibidores de Proteases/farmacologia , Dissulfetos , PapaínaRESUMO
Among the twelve catalytically active carbonic anhydrase isozymes present in the human body, the CAIX is highly overexpressed in various solid tumors. The enzyme acidifies the tumor microenvironment enabling invasion and metastatic processes. Therefore, many attempts have been made to design chemical compounds that would exhibit high affinity and selective binding to CAIX over the remaining eleven catalytically active CA isozymes to limit undesired side effects. It has been postulated that such drugs may have anticancer properties and could be used in tumor treatment. Here we have designed a series of compounds, methyl 5-sulfamoyl-benzoates, which bear a primary sulfonamide group, a well-known marker of CA inhibitors, and determined their affinities for all twelve CA isozymes. Variations of substituents on the benzenesulfonamide ring led to compound 4b, which exhibited an extremely high observed binding affinity to CAIX; the Kd was 0.12 nM. The intrinsic dissociation constant, where the binding-linked protonation reactions have been subtracted, reached 0.08 pM. The compound also exhibited more than 100-fold selectivity over the remaining CA isozymes. The X-ray crystallographic structure of compound 3b bound to CAIX showed the structural position, while several structures of compounds bound to other CA isozymes showed structural reasons for compound selectivity towards CAIX. Since this series of compounds possess physicochemical properties suitable for drugs, they may be developed for anticancer therapeutic purposes.
Assuntos
Benzoatos/farmacologia , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Sulfonamidas/farmacologia , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X/métodos , Humanos , Isoenzimas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Ligação Proteica/fisiologia , Relação Estrutura-Atividade , Termodinâmica , Microambiente Tumoral/efeitos dos fármacos , BenzenossulfonamidasRESUMO
The human genome encodes two highly similar cytosolic Hsp90 proteins called isoforms Hsp90α and Hsp90ß. Of the 300 client proteins for Hsp90 identified so far only a handful interact specifically with one Hsp90 isoform. Here we report for the first time that Hsp90 cochaperone p23 binds preferentially to Hsp90α and that this interaction is mediated by the middle domain of Hsp90α. Based on the homology modeling, we infer that the middle domains in the Hsp90α dimer bind stronger with each other than in the Hsp90ß dimer. Therefore, compared to Hsp90ß, Hsp90α may adopt closed conformation more easily. Hsp90 interacts with p23 in the closed conformation. Hsp90α binds human recombinant p23 about three times stronger than Hsp90ß but with significantly smaller exothermic enthalpy as determined by isothermal titration calorimetry of direct binding between the purified proteins. As p23 binds to Hsp90 in a closed conformation, stabilization of the Hsp90α dimer in the closed conformation by its middle domains explains preference of p23 to this Hsp90 isoform.
Assuntos
Proteínas de Choque Térmico HSP90/química , Chaperonas Moleculares/química , Células Cultivadas , Dimerização , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Conformação ProteicaRESUMO
Membrane-associated carbonic anhydrase (CA) isoform IV participates in carbon metabolism and pH homeostasis and is implicated in the development of eye diseases such as retinitis pigmentosa and glaucoma. A series of substituted benzenesulfonamides were designed and their binding affinity to CA IV was determined by fluorescent thermal shift assay and isothermal titration calorimetry (ITC). Compound [(4-chloro-2-phenylsulfanyl-5-sulfamoyl-benzoyl)amino]propyl acetate (19) bound CA IV with the K d of 1.0 nM and exhibited significant selectivity over the remaining 11 human CA isoforms. The compound could be developed as a drug targeting CA IV. Various forms of recombinant CA IV were produced in Escherichia coli and mammalian cell cultures. Comparison of their temperature stability in various buffers and salt solutions demonstrated that CA IV is most stable at slightly alkaline conditions and at elevated sodium sulfate concentrations. High-resolution X-ray crystallographic structures of ortho-Cl and meta-thiazole-substituted benzene sulfonamide in complex with CA IV revealed the position of and interactions between the ligand and the protein. Sulfonamide inhibitor binding to CA IV is linked to several reactions-the deprotonation of the sulfonamide amino group, the protonation of CA-Zn(II)-bound hydroxide at the active site of CA IV, and the compensating reactions of the buffer. The dissection of binding-linked reactions yielded the intrinsic thermodynamic parameters, characterizing the interaction between CA IV and the sulfonamides in the binding-able protonation forms, including Gibbs energy, enthalpy, and entropy, that could be used for the characterization of binding to any CA in the process of drug design.
Assuntos
Anidrase Carbônica IV/antagonistas & inibidores , Anidrase Carbônica IV/metabolismo , Inibidores da Anidrase Carbônica/metabolismo , Proteínas Recombinantes/metabolismo , Anidrase Carbônica IV/química , Inibidores da Anidrase Carbônica/farmacologia , Domínio Catalítico , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Proteínas Recombinantes/química , TermodinâmicaRESUMO
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.
Assuntos
Anidrase Carbônica IX , Inibidores da Anidrase Carbônica , Sulfonamidas , Sulfonamidas/química , Inibidores da Anidrase Carbônica/química , Humanos , Anidrase Carbônica IX/metabolismo , Anidrase Carbônica IX/antagonistas & inibidores , Anidrase Carbônica IX/química , Ligação Proteica , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/química , Concentração de Íons de HidrogênioRESUMO
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.
Assuntos
Desenho de Fármacos , Proteínas de Choque Térmico HSP90 , Imidazóis , Resorcinóis , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Resorcinóis/química , Resorcinóis/farmacologia , Resorcinóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/síntese química , Relação Estrutura-Atividade , Estrutura Molecular , Relação Dose-Resposta a Droga , Simulação de Dinâmica Molecular , Simulação de Acoplamento Molecular , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese químicaRESUMO
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/.
Assuntos
Inibidores da Anidrase Carbônica , Anidrases Carbônicas , Humanos , Ligantes , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/metabolismo , Termodinâmica , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Ligação ProteicaRESUMO
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.
RESUMO
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.
Assuntos
Anidrases Carbônicas , Neoplasias , Humanos , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Corantes Fluorescentes , Anidrases Carbônicas/metabolismo , Linhagem Celular Tumoral , Antígenos de Neoplasias/metabolismo , Sulfonamidas/farmacologia , FluoresceínasRESUMO
A key part of the optimization of small molecules in pharmaceutical inhibitor development is to vary the molecular design to enhance complementarity of chemical features of the compound with the positioning of amino acids in the active site of a target enzyme. Typically this involves iterations of synthesis, to modify the compound, and biophysical assay, to assess the outcomes. Selective targeting of the anti-cancer carbonic anhydrase isoform XII (CA XII), this process is challenging because the overall fold is very similar across the twelve CA isoforms. To enhance drug development for CA XII we used a reverse engineering approach where mutation of the key six amino acids in the active site of human CA XII into the CA II isoform was performed to provide a protein chimera (chCA XII) which is amenable to structure-based compound optimization. Through determination of structural detail and affinity measurement of the interaction with over 60 compounds we observed that the compounds that bound CA XII more strongly than CA II, switched their preference and bound more strongly to the engineered chimera, chCA XII, based on CA II, but containing the 6 key amino acids from CA XII, behaved as CA XII in its compound recognition profile. The structures of the compounds in the chimeric active site also resembled those determined for complexes with CA XII, hence validating this protein engineering approach in the development of new inhibitors.
Assuntos
Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/metabolismo , Quimera/metabolismo , Sulfonamidas/química , Amidas/química , Sequência de Aminoácidos , Inibidores da Anidrase Carbônica/metabolismo , Domínio Catalítico , Cristalização , Desenho de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Proteínas Mutantes , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Relação Estrutura-Atividade , Sulfonamidas/farmacologiaRESUMO
Human carbonic anhydrase IX (CA IX) is highly expressed in tumor tissues, and its selective inhibition provides a potential target for the treatment of numerous cancers. Development of potent, highly selective inhibitors against this target remains an unmet need in anticancer therapeutics. A series of fluorinated benzenesulfonamides with substituents on the benzene ring was designed and synthesized. Several of these exhibited a highly potent and selective inhibition profile against CA IX. Three fluorine atoms significantly increased the affinity by withdrawing electrons and lowering the pKa of the benzenesulfonamide group. The bulky ortho substituents, such as cyclooctyl or even cyclododecyl groups, fit into the hydrophobic pocket in the active site of CA IX but not CA II, as shown by the compound's co-crystal structure with chimeric CA IX. The strongest inhibitor of recombinant human CA IX's catalytic domain in human cells achieved an affinity of 50 pM. However, the high affinity diminished the selectivity. The most selective compound for CA IX exhibited 10 nM affinity. The compound that showed the best balance between affinity and selectivity bound with 1 nM affinity. The inhibitors described in this work provide the basis for novel anticancer therapeutics targeting CA IX.