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1.
J Inorg Biochem ; 256: 112547, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38581802

RESUMO

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 Hidrogênio
2.
Phys Chem Chem Phys ; 26(11): 8767-8774, 2024 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-38420672

RESUMO

Carbonic anhydrase IX (CA IX) is a subtype of the human carbonic anhydrase (hCA) family and exhibits high expression in various solid tumors, rendering it a promising target for tumor therapy. Currently, marketed carbonic anhydrase inhibitors (CAIs) are primarily composed of sulfonamides derivatives, which may have impeded their potential for further expansion. Therefore, we have developed a structure-based virtual screening approach to explore novel CAIs exhibiting distinctive structures and anti-tumor potential in the FDA database. In vitro experiments demonstrated that 3-pyridinemethanol (0.42 µM), procodazole (8.35 µM) and pamidronic acid (8.51 µM) exhibited inhibitory effects on CA IX activity. The binding stability and interaction mode between the CA IX and the hit compounds are further investigated through molecular dynamics simulations and binding free energy calculations. Furthermore, the ADME/Tox prediction results indicated that these compounds exhibited favorable pharmacological properties and minimal toxic side effects. Our study successfully applied computational strategies to discover three non-sulfonamide inhibitors of carbonic anhydrase IX (CA IX) that demonstrate inhibitory activity in vitro. These findings have significant implications for the development of CA IX inhibitors and anti-tumor drugs, contributing to their progress in the field.


Assuntos
Anidrases Carbônicas , Neoplasias , Humanos , Anidrase Carbônica IX/química , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Relação Estrutura-Atividade , Anidrases Carbônicas/metabolismo , Anidrases Carbônicas/uso terapêutico , Neoplasias/tratamento farmacológico , Sulfonamidas/química , Sulfanilamida , Estrutura Molecular
3.
MAbs ; 15(1): 2248672, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37622732

RESUMO

Carbonic anhydrase (CA)-IX is an extracellular enzyme that is essential in the adaptation of tumor cells to their increasingly more hypoxic and acidic microenvironment. Within the family of carbonic anhydrases, CA-IX is unique in that it is the only CA with an N-terminal intrinsically disordered region (IDR) containing a proteoglycan (PG)-like domain. This PG-like IDR has been described to be instrumental in CA-IX's enzyme activity, as well as tumor cell motility and invasion. We have characterized the antibody-epitope interactions of two novel and unique antibodies (11H9 and 12H8) that are specific for the human CA-IX's IDR. Binding interactions of these antibodies to the intact IDR were studied by surface plasmon resonance and high-resolution nuclear magnetic resonance (NMR) spectroscopy, while the specific epitopes were determined by both NMR and yeast surface display (YSD). Our data show that 12H8 binds to the N-terminus of CA-IX, while 11H9 has a high affinity for an epitope located in the central region of the IDR containing three GEEDLP repeats in a manner that is different from the previously described M75 antibody. Titration NMR spectroscopy using CA-IX's entire IDR in addition identified a secondary epitope of 11H9 at the beginning of the PG-like domain that remains exposed and available for further binding events after the engagement at its primary epitope at the center of the PG-like domain. Transverse relaxation optimized NMR spectroscopy of 11H9-F(Ab) in complex with the CA-IX IDR outlines structural rigidification of a linear epitope, while the rest of the IDR remains largely unstructured upon complex formation. This study illustrates how high-resolution NMR and YSD are used as complementary tools for a comprehensive characterization of antibody-epitope interactions involving intrinsically unstructured antigen domains with highly repetitive sequences.


Assuntos
Anidrases Carbônicas , Saccharomyces cerevisiae , Humanos , Anidrase Carbônica IX/química , Anidrase Carbônica IX/metabolismo , Saccharomyces cerevisiae/metabolismo , Epitopos , Proteoglicanas , Antígenos de Neoplasias , Anidrases Carbônicas/química , Espectroscopia de Ressonância Magnética
4.
Arch Pharm (Weinheim) ; 356(10): e2300316, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37495909

RESUMO

Carbonic anhydrase isoforms IX and XII are overexpressed in hypoxic tumor cells regulating various physiological processes such as cell proliferation, invasion, and metastasis, resulting in the onset and spread of cancer. Selective inhibition of these enzymes is a promising strategy for anticancer therapy. Coumarin derivatives were identified as selective and potent inhibitors of these isoforms. This study reports 6-aminocoumarin sulfonamide and oxime ether derivatives linked through a chloroacetyl moiety tethered to piperazine and piperidone, respectively, showing selective inhibition against human carbonic anhydrase (hCA) IX and XII with Ki ranging from 0.51 to 1.18 µM and 0.89-4.43 µM. While the sulfonamide derivative 8a exhibited submicromolar inhibition against hCA IX and XII with Ki 0.89 and 0.51 µM, the oxime ether derivatives showed lower activity than the sulfonamides, with the compound 5n inhibiting hCA IX and hCA XII with a Ki of 1.055 and 0.70 µM, respectively. The above results demonstrate the potential of these derivatives as selective, potent inhibitors of carbonic anhydrase IX and XII and provide a foundation for further optimization and development as effective anticancer agents. Further, the binding mode of the synthesized derivatives in the active site were examined using molecular docking and dynamic simulation studies.


Assuntos
Anidrases Carbônicas , Simulação de Dinâmica Molecular , Humanos , Anidrase Carbônica IX/química , Anidrase Carbônica IX/metabolismo , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Éter , Sulfonamidas/química , Anidrases Carbônicas/metabolismo , Cumarínicos/farmacologia , Cumarínicos/química , Etil-Éteres , Éteres , Isoformas de Proteínas/metabolismo , Inibidores da Anidrase Carbônica , Estrutura Molecular
5.
J Biomol Struct Dyn ; 41(24): 15243-15261, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36914238

RESUMO

All the previously reported phenylpyrazoles as carbonic anhydrase inhibitors (CAIs) were found to have small sizes and high levels of flexibility, and hence showed low selectivity profiles toward a particular isoform of CA. Herein, we report the development of a more rigid ring system bearing a sulfonamide hydrophilic head and a lipophilic tail to develop novel molecules that are suggested to have a better selectivity toward a special CA isoform. Accordingly, three novel sets of pyrano[2,3-c]pyrazoles attached with sulfonamide head and aryl hydrophobic tail were synthesized to enhance the selectivity toward a specific isoform of human carbonic anhydrases (hCAs). The impact of both attachments on the potency and selectivity has been extensively discussed in terms of in vitro cytotoxicity evaluation under hypoxic conditions, structure-activity relationship and carbonic anhydrase enzyme assay. All of the new candidates displayed good cytotoxic activities against breast and colorectal carcinomas. Results of the carbonic anhydrase enzyme assay demonstrated the preferential of compounds 22, 24 and 27 to inhibit the isoform IX of hCAs selectively. Wound-healing assay has also been performed and revealed the potential of 27 to decrease the wound closure percentage in MCF-7 cells. Molecular docking and molecular orbital analysis have finally been conducted. Results indicate the potential binding interactions of 24 and 27 with several crucial amino acids of the hCA IX.Communicated by Ramaswamy H. Sarma.


Assuntos
Anidrases Carbônicas , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Anidrase Carbônica IX/química , Relação Estrutura-Atividade , Sulfonamidas/química , Isoformas de Proteínas/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/química
6.
Arch Pharm (Weinheim) ; 356(4): e2200508, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36587981

RESUMO

Currently, cancer is the most grieving threat to society. The cancer-related death rate has had an ascending trend, despite the implementation of numerous treatment strategies or the discovery of an array of potent molecules against several pathways of cancer growth. The need of the hour is to prevent the multidrug resistance toll, and the current efforts have been bestowed upon a versatile small molecule scaffold, coumarin (benz[α]pyrone), a natural compound possessing interesting affinity toward the cancer target human carbonic anhydrase (hCA), focusing on hCA I, II, IX, and XII. Along with coumarin, the age-old known antibacterial drug sulfonamide, when conjugated at positions 3, 7, and 8 of coumarin either with a linker group or as a single entity, has been reported to enhance the affinity of coumarin toward the overexpressed enzymes in tumor cell lines. The sulfonamides have been listed as obsolete drugs due to the severe side effects caused by them; however, their affinity toward the hCA-zinc-binding core has attracted the attention of researchers. Hence, in the process of drug development, coumarin and sulfonamides have remained the choice of last resort. To unveil the synthetic strategy of coumarin-sulfonamide conjugation, their rationale for inhibiting cancer cells/enzymes, and their affinity toward various types of carcinoma have been the sole goal of the researchers. This review specifically focuses on the mechanism of action and the structure-activity relationship through synthetic strategies and the binding affinity of coumaryl-sulfonamide conjugates with the anticancer targets possessing the highest enzyme affinity, since 2008.


Assuntos
Antineoplásicos , Anidrases Carbônicas , Humanos , Relação Estrutura-Atividade , Anidrase Carbônica IX/química , Anidrase Carbônica IX/metabolismo , Estrutura Molecular , Anidrases Carbônicas/metabolismo , Desenvolvimento de Medicamentos , Antineoplásicos/farmacologia , Antineoplásicos/química , Cumarínicos/farmacologia , Cumarínicos/química , Sulfonamidas/farmacologia , Sulfonamidas/química , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/química
7.
Oncol Rep ; 49(2)2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36524367

RESUMO

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme participating in adaptive responses of tumors to hypoxia and acidosis. CA IX regulates pH, facilitates metabolic reprogramming, and supports migration, invasion and metastasis of cancer cells. Extracellular domain (ECD) of CA IX can be shed to medium and body fluids by a disintegrin and metalloproteinase (ADAM) 17. Here we show for the first time that CA IX ECD shedding can be also executed by ADAM10, a close relative of ADAM17, via an overlapping cleavage site in the stalk region of CA IX connecting its exofacial catalytic site with the transmembrane region. This finding is supported by biochemical evidence using recombinant human ADAM10 protein, colocalization of ADAM10 with CA IX, ectopic expression of a dominant­negative mutant of ADAM10 and RNA interference­mediated suppression of ADAM10. Induction of the CA IX ECD cleavage with ADAM17 and/or ADAM10 activators revealed their additive effect. Similarly, additive effect was observed with an ADAM17­inhibiting antibody and an ADAM10­preferential inhibitor GI254023X. These data indicated that ADAM10 is a CA IX sheddase acting on CA IX non­redundantly to ADAM17.


Assuntos
Proteínas ADAM , Anidrase Carbônica IX , Humanos , Proteínas ADAM/química , Proteínas ADAM/metabolismo , Proteína ADAM10/química , Proteína ADAM10/metabolismo , Proteína ADAM17/química , Proteína ADAM17/metabolismo , Anidrase Carbônica IX/química , Anidrase Carbônica IX/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo
8.
Int J Mol Sci ; 23(22)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36430173

RESUMO

It is postulated that the overexpression of Carbonic Anhydrase isozyme IX in some cancers contributes to the acidification of the extracellular matrix. It was proved that this promotes the growth and metastasis of the tumor. These observations have made Carbonic Anhydrase IX an attractive drug target. In the light of the findings and importance of the glycoprotein in the cancer treatment, we have employed quantum-chemical approaches to study non-covalent interactions in the binding pocket. As a ligand, the acetazolamide (AZM) molecule was chosen, being known as a potential inhibitor exhibiting anticancer properties. First-Principles Molecular Dynamics was performed to study the chalcogen and other non-covalent interactions in the AZM ligand and its complexes with amino acids forming the binding site. Based on Density Functional Theory (DFT) and post-Hartree-Fock methods, the metric and electronic structure parameters were described. The Non-Covalent Interaction (NCI) index and Atoms in Molecules (AIM) methods were applied for qualitative/quantitative analyses of the non-covalent interactions. Finally, the AZM-binding pocket interaction energy decomposition was carried out. Chalcogen bonding in the AZM molecule is an important factor stabilizing the preferred conformation. Free energy mapping via metadynamics and Path Integral molecular dynamics confirmed the significance of the chalcogen bond in structuring the conformational flexibility of the systems. The developed models are useful in the design of new inhibitors with desired pharmacological properties.


Assuntos
Calcogênios , Neoplasias , Humanos , Anidrase Carbônica IX/química , Ligantes , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/química , Acetazolamida/farmacologia , Acetazolamida/química , Calcogênios/química
9.
Molecules ; 27(14)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35889480

RESUMO

A novel series of thiosemicarbazide-substituted coumarins was synthesized and the inhibitory effects against four physiologically relevant carbonic anhydrase isoforms I, II, IX and XII showed selective activities on the tumor-associated IX and XII isozymes. Molecular modeling studies on selected compounds 14a and 22a were performed. The binding modes of such compounds were determined assuming their enzymatically active structures (i.e., cinnamic acid) in the thermodynamically favored, and not previously explored, E geometry. Molecular modelling suggests multiple interactions within the enzymatic cavity and may explain the high potency and selectivity reported for the hCAs IX and XII.


Assuntos
Anidrases Carbônicas , Neoplasias , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica I , Anidrase Carbônica IX/química , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/química , Cumarínicos/química , Cumarínicos/farmacologia , Humanos , Estrutura Molecular , Neoplasias/tratamento farmacológico , Semicarbazidas , Relação Estrutura-Atividade
10.
J Biomol Struct Dyn ; 40(10): 4516-4531, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-33317405

RESUMO

Human carbonic anhydrase IX (hCA IX) is a promising target for the development of potential anticancer agents. In the current study, pharmacophore and 3D-QSAR models have been developed using SLC-0111 derivatives. The developed models have been further utilized for the virtual screening process to develop potent hCA IX inhibitors. Thirteen different models have been developed by employing various combinations of training and test set molecules. Based on this, a model, AADDR.135, comprising two H-bond acceptors, two H-bond donors and one aromatic ring, has been found as the best QSAR model. The proposed model exhibits high robustness (R2 = 0.9789), with good predictive ability (Q2 = 0.6872). An external library of drug-like compounds (∼10000 molecules) imported from the ZINC15 database has been screened over the model AADDR.135. In total, 1601 compounds were obtained as hits. Molecular docking studies and molecular dynamics simulations have been performed on the obtained hits and, based on these computations, two unique molecules have been identified as potential hCA IX inhibitors. These show higher binding energies compared to the parent molecule and its most potent analogue.Communicated by Ramaswamy H. Sarma.


Assuntos
Simulação de Dinâmica Molecular , Relação Quantitativa Estrutura-Atividade , Anidrase Carbônica IX/química , Humanos , Simulação de Acoplamento Molecular
11.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445506

RESUMO

Hypoxia-regulated protein carbonic anhydrase IX (CA IX) is up-regulated in different tumor entities and correlated with poor prognosis in breast cancer patients. Due to the radio- and chemotherapy resistance of solid hypoxic tumors, derivatives of betulinic acid (BA), a natural compound with anticancer properties, seem to be promising to benefit these cancer patients. We synthesized new betulin sulfonamides and determined their cytotoxicity in different breast cancer cell lines. Additionally, we investigated their effects on clonogenic survival, cell death, extracellular pH, HIF-1α, CA IX and CA XII protein levels and radiosensitivity. Our study revealed that cytotoxicity increased after treatment with the betulin sulfonamides compared to BA or their precursors, especially in triple-negative breast cancer (TNBC) cells. CA IX activity as well as CA IX and CA XII protein levels were reduced by the betulin sulfonamides. We observed elevated inhibitory efficiency against protumorigenic processes such as proliferation and clonogenic survival and the promotion of cell death and radiosensitivity compared to the precursor derivatives. In particular, TNBC cells showed benefit from the addition of sulfonamides onto BA and revealed that betulin sulfonamides are promising compounds to treat more aggressive breast cancers, or are at the same level against less aggressive breast cancer cells.


Assuntos
Antineoplásicos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Triterpenos Pentacíclicos/química , Sulfonamidas/farmacologia , Neoplasias de Mama Triplo Negativas/metabolismo , Antígenos de Neoplasias/química , Antineoplásicos/síntese química , Antineoplásicos/química , Anidrase Carbônica IX/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Simulação de Acoplamento Molecular , Tolerância a Radiação , Sulfonamidas/síntese química , Sulfonamidas/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ácido Betulínico
12.
J Biol Chem ; 296: 100505, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33675747

RESUMO

Low levels of oxygen (hypoxia) occurs in many (patho)physiological situations. Adaptation to hypoxia is in part mediated by proteins expressed in the extracellular space that mature in the endoplasmic reticulum (ER) prior to traversing the secretory pathway. The majority of such ER cargo proteins require disulfide bonds for structural stability. Disulfide bonds are formed co- and posttranslationally in a redox relay that requires a terminal electron acceptor such as oxygen. We have previously demonstrated that some ER cargo proteins such as low-density lipoprotein receptor (LDLR) and influenza hemagglutinin (Flu-HA) are unable to complete disulfide bond formation in the absence of oxygen, limiting their ability to pass ER quality control and their ultimate expression. Here, using radioactive pulse-chase immunoprecipitation analysis, we demonstrate that hypoxia-induced ER cargo proteins such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A) complete disulfide bond formation and mature with similar kinetics under hypoxia and normoxia. A global in silico analysis of ER cargo revealed that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other ER cargo proteins. These data demonstrate the existence of alternative electron acceptors to oxygen for disulfide bond formation in cellulo. However, the ability of different proteins to utilize an oxygen-independent pathway for disulfide bond formation varies widely, contributing to differential gene expression in hypoxia. The superior ability of hypoxia-induced proteins such as VEGF-A and CA9 to mature in hypoxia may be conferred by a simpler disulfide architecture.


Assuntos
Antígenos de Neoplasias/química , Anidrase Carbônica IX/química , Hipóxia Celular , Dissulfetos/química , Retículo Endoplasmático/metabolismo , Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX/metabolismo , Dissulfetos/metabolismo , Células HeLa , Humanos , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Cell Mol Life Sci ; 78(5): 2059-2067, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33201250

RESUMO

hCA IX is a multi-domain protein belonging to the family of hCAs which are ubiquitous zinc enzymes that catalyze the reversible hydration of CO2 to HCO3- and H+. hCA IX is a tumor-associated enzyme with a limited distribution in normal tissues, but over-expressed in many tumors, and is a promising drug target. Although many studies concerning the CA IX catalytic domain were performed, little is known about the proteoglycan-like (PG-like) domain of hCA IX which has been poorly investigated so far. Here we attempt to fill this gap by providing an overview on the functional, structural and therapeutic studies of the PG-like domain of hCA IX which represents a unique feature within the CA family. The main studies and recent advances concerning PG role in modulating hCA IX catalytic activity as well as in tumor spreading and migration are here reported. Special attention has been paid to the newly discovered disordered features of the PG domain which open new perspectives about its molecular mechanisms of action under physiological and pathological conditions, since disorder is likely involved in mediating interactions with partner proteins. The emerged disordered features of PG domain will be explored for putative diagnostic and therapeutic applications involving CA IX targeting in tumors.


Assuntos
Anidrase Carbônica IX/metabolismo , Domínio Catalítico , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteoglicanas/metabolismo , Bicarbonatos/metabolismo , Biocatálise/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Anidrase Carbônica IX/química , Inibidores da Anidrase Carbônica/farmacologia , Inibidores da Anidrase Carbônica/uso terapêutico , Concentração de Íons de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Proteoglicanas/química
14.
Bioorg Chem ; 103: 104204, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32891000

RESUMO

Building on the conclusions of previous inhibition studies with pyridinium-benzenesulfonamides from our team and on the X-ray crystal structure of the lead compound identified, a series of 24 pyridinium derivatives of 3-aminobenzenesulfonamide was synthesized and investigated for carbonic anhydrase inhibition. The new pyridinium-sulfonamides were evaluated as inhibitors of four human carbonic anhydrase (CA, EC 4.2.1.1) isoforms, namely CA I, CA II (cytosolic), CA IX and XII (transmembrane, tumor-associated forms). Excellent inhibitory activity in the nanomolar range was observed against CA IX with most of these sulfonamides, and against CA XII (nanomolar/sub-nanomolar) with some of the new compounds. These sulfonamides were generally potent inhibitors of CA II and CA I too. Docking studies revealed a preference of these compounds to bind the P1 hydrophobic site of CAs, supporting the observed inhibition profile. The salt-like nature of these positively charged sulfonamides can further focus the inhibitory ability on membrane-bound CA IX and CA XII and could efficiently decrease the viability of three human carcinomas under hypoxic conditions where these isozymes are over-expressed, thus recommending the new compounds as potential diagnostic tools or therapeutic agents.


Assuntos
Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/metabolismo , Neoplasias/enzimologia , Compostos de Piridínio/farmacologia , Sulfonamidas/farmacologia , Antígenos de Neoplasias/química , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Sequência de Bases , Anidrase Carbônica IX/química , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/química , Domínio Catalítico , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Compostos de Piridínio/síntese química , Compostos de Piridínio/metabolismo , Sulfonamidas/síntese química , Sulfonamidas/metabolismo
15.
Chemistry ; 26(69): 16541-16553, 2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-32757220

RESUMO

Carbonic anhydrase IX (CA IX), a tumor-associated metalloenzyme, represents a validated target for cancer therapy and diagnostics. Herein, we report the inhibition properties of isomeric families of sulfonamidopropyl-dicarba-closo-dodecaboranes group(s) prepared using a new direct five-step synthesis from the corresponding parent cages. The protocol offers a reliable solution for synthesis of singly and doubly substituted dicarba-closo-dodecaboranes with a different geometric position of carbon atoms. The closo-compounds from the ortho- and meta-series were then degraded to corresponding 11-vertex dicarba-nido-undecaborate(1-) anions. All compounds show in vitro enzymatic activity against CA IX in the low nanomolar or subnanomolar range. This is accompanied by clear isomer dependence of the inhibition constant (Ki ) and selectivity towards CA IX. Decreasing trends in Ki and selectivity index (SI ) values are observed with increasing separation of the cage carbon atoms. Interactions of compounds with the active sites of CA IX were explored with X-ray crystallography, and eight high-resolution crystal structures uncovered the structural basis of inhibition potency and selectivity.


Assuntos
Antígenos de Neoplasias/química , Anidrase Carbônica IX/química , Anidrase Carbônica I/química , Inibidores da Anidrase Carbônica , Neoplasias , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica I/metabolismo , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Humanos , Isoenzimas , Relação Estrutura-Atividade
16.
Eur J Med Chem ; 201: 112478, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32659606

RESUMO

This paper describes identification of the first-in-class multi-target adenosine A2A receptor antagonists-carbonic anhydrase (CA) IX and XII inhibitors, as new potential antitumor agents. To obtain the multi-acting ligands, the 8-amino-2,6-diphenyltriazolo[4,3-a]pyrazin-3-one, a potent adenosine hA2A receptor (AR) antagonist, was taken as lead compound. To address activity against the tumor-associated CA isoforms, it was modified by introduction of different substituents (OH, COOH, CONHOH, SO2NH2) on the 6-phenyl ring or on a phenyl pendant connected to the former through different spacers. Among the new triazolopyrazines 1-23, the most active were those featuring the sulfonamide residue. Derivative 20, featuring a 4-sulfonamidophenyl residue attached through a CONH(CH2)2CONH spacer at the para-position of the 6-phenyl ring, showed the best combination of activity at the three targets. In fact, it inhibited both the tumor-associated hCA IX and XII isozymes at nanomolar concentration (Ki = 5.0 and 27.0 nM), and also displayed a quite good affinity for the hA2A AR (Ki = 108 nM). Compound 14, bearing the 4-sulfonamidophenyl residue linked at the para-position of the 6-phenyl ring by a CONH spacer, was remarkable because both its hA2A AR affinity and hCA XII inhibitory potency were in the low nanomolar range (Ki = 6.4 and 6.2 nM, respectively). Molecular docking studies highlighted the interaction mode of selected triazolopyrazines in the hA2A AR recognition pocket and in the active site of hCA II, IX and XII isoforms.


Assuntos
Antagonistas do Receptor A2 de Adenosina/química , Antineoplásicos/química , Inibidores da Anidrase Carbônica/química , Pirazinas/química , Triazóis/química , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/metabolismo , Animais , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Células CHO , Anidrase Carbônica IX/química , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/química , Anidrases Carbônicas/metabolismo , Domínio Catalítico , Cricetulus , Ensaios Enzimáticos , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Pirazinas/síntese química , Pirazinas/metabolismo , Receptor A2A de Adenosina/metabolismo , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/metabolismo
17.
Int J Mol Sci ; 21(15)2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32722392

RESUMO

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.


Assuntos
Antígenos de Neoplasias/química , Anidrase Carbônica IX/química , Proteínas de Neoplasias/química , Neoplasias/enzimologia , Cristalografia por Raios X , Humanos , Domínios Proteicos
18.
J Am Chem Soc ; 142(28): 12333-12340, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32539375

RESUMO

Dimeric proteins are prominent in biology, and receptor dimerization (homo- or heterodimerization) is central to signal transduction. Herein, we report a network that responds to a membrane-associated dimeric protein with the uncaging of a powerful cytotoxic. The network is based on two ligands functionalized with peptide nucleic acids (PNAs) (templating strand and catalyst-functionalized strand, respectively) and a substrate with the caged cytotoxic (monomethyl auristatin E: MMAE; a high-affinity tubulin ligand). In the presence of the dimeric protein, the network yields a cooperative supramolecular assembly with a hybridization architecture that enhances the templated reaction and enables the uncaging of a substrate. The network was tested on cells that express a cancer biomarker, carbonic anhydrase IX, in response to hypoxia. The output of the network correlates with the expression of carbonic anhydrase IX, and this biomarker was harnessed to uncage a potent cytotoxic agent.


Assuntos
Antígenos de Neoplasias/química , Biomarcadores Tumorais/química , Anidrase Carbônica IX/química , Proteínas de Membrana/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Anidrase Carbônica IX/genética , Anidrase Carbônica IX/metabolismo , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estrutura Molecular , Oligopeptídeos/farmacologia , Ácidos Nucleicos Peptídicos/farmacologia , Células Tumorais Cultivadas
19.
Int J Mol Sci ; 21(10)2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32456080

RESUMO

Various sulfonamide derivatives are intensively studied as anticancer agents owing to their inhibitory activity against human tumor-associated carbonic anhydrase isoforms. In this work, different synthetic procedures for the series of 1,3,5-triazinyl-aminobenzenesulfonamide conjugates with amino acids, possessing polar uncharged, negatively charged, and hydrophobic side chain, were studied and optimized with respect to the yield/purity of the synthesis/product as well as the time of synthetic reaction. These procedures were compared to each other via characteristic HPLC-ESI-DAD/QTOF/MS analytical product profiles, and their benefits as well as limitations were discussed. For new sulfonamide derivatives, incorporating s-triazine with a symmetric pair of polar and some less-polar proteinogenic amino acids, inhibition constants (KIs) against four human carboanhydrases (hCAs), namely cytosolic hCA I, II, transmembrane hCA IV, and the tumor-associated, membrane-bound hCA IX isoforms, were computationally predicted applying various methods of the advanced statistical analysis. Quantitative structure-activity relationship (QSAR) analysis indicated an impressive KI ratio (hCA II/hCA IX) 139.1 and hCA IX inhibition constant very similar to acetazolamide (KI = 29.6 nM) for the sulfonamide derivative disubstituted with Gln. The derivatives disubstituted with Ser, Thr, and Ala showed even lower KIs (8.7, 13.1, and 8.4 nM, respectively).


Assuntos
Aminoácidos/química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/química , Sulfonamidas/química , Triazinas/química , Acetazolamida/farmacologia , Antígenos de Neoplasias/química , Anidrase Carbônica I/química , Anidrase Carbônica II/química , Anidrase Carbônica IV/química , Anidrase Carbônica IX/química , Inibidores da Anidrase Carbônica/farmacologia , Cromatografia Líquida de Alta Pressão , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas , Modelos Moleculares , Redes Neurais de Computação , Isoformas de Proteínas/metabolismo , Software , Relação Estrutura-Atividade , Benzenossulfonamidas
20.
Angew Chem Int Ed Engl ; 59(26): 10456-10460, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32150656

RESUMO

The development of nanoparticle-based biomedical applications has been hampered due to undesired off-target effects. Herein, we outline a cellular AND gate to enhance uptake selectivity, in which a nanoassembly-cell interaction is turned on, only in the concurrent presence of two different protein functions, an enzymatic reaction (alkaline phosphatase, ALP) and a ligand-protein (carbonic anhydrase IX, CA IX) binding event. Selective uptake of nanoassemblies was observed in cells that overexpress both of these proteins (unicellular AND gate). Interestingly, selective uptake can also be achieved in CA IX overexpressed cells, when cocultured with ALP overexpressed cells, where the nanoassembly presumably acts as a mediator for cell-cell communication (bicellular AND gate). This logic-gated cellular uptake could find use in applications such as tumor imaging or theranostics.


Assuntos
Fosfatase Alcalina/metabolismo , Anidrase Carbônica IX/metabolismo , Corantes Fluorescentes/metabolismo , Nanopartículas/metabolismo , Fosfatase Alcalina/química , Animais , Anidrase Carbônica IX/química , Bovinos , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Humanos , Nanopartículas/química , Organofosfatos/química , Organofosfatos/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Ácidos Polimetacrílicos/química , Ácidos Polimetacrílicos/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo
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