Unlocking the paracrine crosstalk: adipocyte-derived factors affect carbonic anhydrase IX expression in colon and breast cancer cells.

Obesity is a major public health concern because it increases the risk of several diseases, including cancer. Crosstalk between obesity and cancer seems to be very complex, and the interaction between adipocytes and cancer cells leads to changes in adipocytes' function and their paracrine signaling, promoting a microenvironment that supports tumor growth. Carbonic anhydrase IX (CA IX) is a tumor-associated enzyme that not only participates in pH regulation but also facilitates metabolic reprogramming and supports the migration, invasion, and metastasis of cancer cells. In addition, CA IX expression, predominantly regulated via hypoxia-inducible factor (HIF-1), serves as a surrogate marker of hypoxia. In this study, we investigated the impact of adipocytes and adipocyte-derived factors on the expression of CA IX in colon and breast cancer cells. We observed increased expression of CA9 mRNA as well as CA IX protein in the presence of adipocytes and adipocyte-derived conditioned medium. Moreover, we confirmed that adipocytes affect the hypoxia signaling pathway and that the increased CA IX expression results from adipocyte-mediated induction of HIF-1α. Furthermore, we demonstrated that adipocyte-mediated upregulation of CA IX leads to increased migration and decreased adhesion of colon cancer cells. Finally, we brought experimental evidence that adipocytes, and more specifically leptin, upregulate CA IX expression in cancer cells and consequently promote tumor progression.

OTUB2 silencing promotes ovarian cancer via mitochondrial metabolic reprogramming and can be synthetically targeted by CA9 inhibition.

Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.

Can hypoxia marker carbonic anhydrase IX serve as a potential new diagnostic marker and therapeutic target of non-small cell lung cancer?

Lung cancer represents the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is a molecularly heterogeneous disease with intratumoral heterogeneity and a significant mutational burden associated with clinical outcome. Tumor microenvironment (TME) plays a fundamental role in the initiation and progression of primary de novo lung cancer and significantly influences the response of tumor cells to therapy. Hypoxia, an integral part of the tumor microenvironment and a serious clinical phenomenon, is associated with increased genetic instability and a more aggressive phenotype of NSCLC, which correlates with the risk of metastasis. Low oxygen concentration influences all components of TME including the immune microenvironment. Hypoxia-inducible pathway activated in response to low oxygen supply mediates the expression of genes important for the adaptation of tumor cells to microenvironmental changes. A highly active transmembrane hypoxia-induced metalloenzyme - carbonic anhydrase IX (CAIX), as a part of transport metabolon, contributes to the maintenance of intracellular pH within physiological values and to the acidification of the extracellular space. CAIX supports cell migration and invasion and plays an important role in NSCLC tumor tissue and pleural effusion. Due to its high expression, it also represents a potential diagnostic differential biomarker and therapeutic target in NSCLC. To test new potential targeted therapeutic compounds, suitable models are required that more faithfully simulate tumor tissue, TME components, and spatial architecture.

Identification of novel CA IX inhibitor: Pharmacophore modeling, docking, DFT, and dynamic simulation.

Human Carbonic anhydrase IX (hCA IX) is found to be an essential biomarker for the treatment of hypoxic tumors in both the early and metastatic stages of cancer. Due to its active function in maintaining pH levels and overexpression in hypoxic conditions, hCA IX inhibitors can be a potential candidate specifically designed to target cancer development at various stages. In search of selective hCA IX inhibitors, we developed a pharmacophore model from the existing natural product inhibitors with IC50 values less than 50 nm. The identified hit molecules were then investigated on protein-ligand interactions using molecular docking experiments followed by molecular dynamics simulations. Among the zinc database 186 hits with an RMSD value less than 1 were obtained, indicating good contact with key residues HIS94, HIS96, HIS119, THR199, and ZN301 required for optimum activity. The top three compounds were subjected to molecular dynamics simulations for 100 ns to know the protein-ligand complex stability. Based on the obtained MD simulation results, binding free energies are calculated. Density Functional Theory (DFT) studies confirmed the energy variation between the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO). The current study has led to the discovery of lead compounds that show considerable promise as hCA IX inhibitors and suggests that three compounds with special molecular features are more likely to be better-inhibiting hCA IX. Compound S35, characterized by a higher stability margin and a smaller energy gap in quantum studies, is an ideal candidate for selective inhibition of CA IX.

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.

Design, synthesis and in vitro evaluation of novel thiazole-coumarin hybrids as selective and potent human carbonic anhydrase IX and XII inhibitors.

The coumarin is one of the most promising classes of non-classical carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. In continuation of our ongoing work on search of coumarin based selective carbonic anhydrase inhibitors, a new series of 6-aminocoumarin based 16 novel analogues of coumarin incorporating thiazole (4a-p) have been synthesized and studied for their hCA inhibitory activity against a panel of human carbonic anhydrases (hCAs). Most of these newly synthesized compounds exhibited interesting inhibition constants in the nanomolar range. Among the tested compounds, the compounds 4f having 4-methoxy substitution exhibited activity at 90.9 nM against hCA XII isoform. It is noteworthy to see that all compounds were specifically and selectively active against isoforms hCA IX and hCA XII, with Ki under 1000 nM range. It is anticipated that these newly synthesized coumarin-thiazole hybrids (4a-p) may emerge as potential leads candidates against hCA IX and hCA XII as selective inhibitors compared to hCA I and hCA II.

Discovery and Mechanistic Studies of Dual-Target Hits for Carbonic Anhydrase IX and VEGFR-2 as Potential Agents for Solid Tumors: X-ray, In Vitro, In Vivo, and In Silico Investigations of Coumarin-Based Thiazoles.

A dual-targeting approach is predicted to yield better cancer therapy outcomes. Consequently, a series of coumarin-based thiazoles (5a-h, 6, and 7a-e) were designed and constructed as potential carbonic anhydrase (CA) and VEGFR-2 suppressors. The inhibitory actions of the target compounds were assessed against CA isoforms IX and VEGFR-2. The assay results showed that coumarin-based thiazoles 5a, 5d, and 5e can effectively inhibit both targets. 5a, 5d, and 5e cytotoxic effects were tested on pancreatic, breast, and prostate cancer cells (PANC1, MCF7, and PC3). Further mechanistic investigation disclosed the ability of 5e to interrupt the PANC1 cell progression in the S stage by triggering the apoptotic cascade, as seen by increased levels of caspases 3, 9, and BAX, alongside the Bcl-2 decline. Moreover, the in vivo efficacy of compound 5e as an antitumor agent was evaluated. Also, molecular docking and dynamics displayed distinctive interactions between 5e and CA IX and VEGFR-2 binding pockets.

Novel Anthraquinone-Based Benzenesulfonamide Derivatives and Their Analogues as Potent Human Carbonic Anhydrase Inhibitors with Antitumor Activity: Synthesis, Biological Evaluation, and In Silico Analysis.

In this study, we designed two series of novel anthraquinone-based benzenesulfonamide derivatives and their analogues as potential carbonic anhydrase inhibitors (CAIs) and evaluated their inhibitory activities against off-target human carbonic anhydrase II (hCA II) isoform and tumor-associated human carbonic anhydrase IX (hCA IX) isoform. Most of these compounds exhibited good inhibitory activities against hCA II and IX. The compounds that exhibited the best hCA inhibition were further studied against the MDA-MB-231, MCF-7, and HepG2 cell lines under hypoxic and normoxic conditions. Additionally, the compounds exhibiting the best antitumor activity were subjected to apoptosis and mitochondrial membrane potential assays, which revealed a significant increase in the percentage of apoptotic cells and a notable decrease in cell viability. Molecular docking studies were performed to demonstrate the presence of numerous hydrogen bonds and hydrophobic interactions between the compounds and the active site of hCA. Absorption, distribution, metabolism, excretion (ADME) predictions showed that all of the compounds had good pharmacokinetic and physicochemical properties.

Preclinical Characterization of DPI-4452: A 68Ga/177Lu Theranostic Ligand for Carbonic Anhydrase IX.

The membrane protein carbonic anhydrase IX (CAIX) is highly expressed in many hypoxic or von Hippel-Lindau tumor suppressor-mutated tumor types. Its restricted expression in healthy tissues makes CAIX an attractive diagnostic and therapeutic target. DPI-4452 is a CAIX-targeting cyclic peptide with a DOTA cage, allowing radionuclide chelation for theranostic purposes. Here, we report CAIX expression in multiple tumor types and provide in vitro and in vivo evaluations of 68Ga-labeled DPI-4452 ([68Ga]Ga-DPI-4452) and 177Lu-labeled DPI-4452 ([177Lu]Lu-DPI-4452). Methods: CAIX expression was assessed by immunohistochemistry with a panel of tumor and healthy tissues. The molecular interactions of complexed and uncomplexed DPI-4452 with CAIX were assessed by surface plasmon resonance and cell-binding assays. In vivo characterization of radiolabeled and nonradiolabeled DPI-4452 was performed in HT-29 colorectal cancer (CRC) and SK-RC-52 clear cell renal cell carcinoma (ccRCC) human xenograft mouse models and in healthy beagle dogs. Results: Overexpression of CAIX was shown in several tumor types, including ccRCC, CRC, and pancreatic ductal adenocarcinoma. DPI-4452 specifically and selectively bound CAIX with subnanomolar affinity. In cell-binding assays, DPI-4452 displayed comparably high affinities for human and canine CAIX but a much lower affinity for murine CAIX, demonstrating that the dog is a relevant species for biodistribution studies. DPI-4452 was rapidly eliminated from the systemic circulation of beagle dogs. The highest uptake of [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 was observed in the small intestine and stomach, 2 organs known to express CAIX. Uptake in other organs (e.g., kidneys) was remarkably low. In HT-29 and SK-RC-52 xenograft mouse models, both [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 showed tumor-selective uptake; in addition, [177Lu]Lu-DPI-4452 significantly reduced tumor growth. These results demonstrated the theranostic potential of DPI-4452. Conclusion: DPI-4452 selectively targets CAIX. [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 localized to tumors and were well tolerated in mice. [177Lu]Lu-DPI-4452 demonstrated strong tumor growth inhibition in 2 xenograft mouse models. Thus, the 2 agents potentially provide a theranostic approach for selecting and treating patients with CAIX-expressing tumors such as ccRCC, CRC, and pancreatic ductal adenocarcinoma.

Discovery of non-sulfonamide carbonic anhydrase IX inhibitors through structure-based virtual screening.

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.

pH regulators and their inhibitors in tumor microenvironment.

As an important characteristic of tumor, acidic tumor microenvironment (TME) is closely related to immune escape, invasion, migration and drug resistance of tumor. The acidity of the TME mainly comes from the acidic products produced by the high level of tumor metabolism, such as lactic acid and carbon dioxide. pH regulators such as monocarboxylate transporters (MCTs), carbonic anhydrase IX (CA IX), and Na+/H+ exchange 1 (NHE1) expel protons directly or indirectly from the tumor to maintain the pH balance of tumor cells and create an acidic TME. We review the functions of several pH regulators involved in the construction of acidic TME, the structure and structure-activity relationship of pH regulator inhibitors, and provide strategies for the development of small-molecule antitumor inhibitors based on these targets.

Tailored Tetrasubstituted Imidazole Carrying the Benzenesulfonamide Fragments as Selective Human Carbonic Anhydrase IX/XII Inhibitors.

A new series of tetrasubstituted imidazole carrying sulfonamide as zinc-anchoring group has been designed. The structures of the synthesized derivatives 5 a-l have been confirmed by spectroscopic analysis. These compounds incorporate an ethylenic spacer between the benzenesulfonamide and the rest of the trisubstituted imidazole moiety and were tested as inhibitors of carbonic anhydrases and for in-vitro cytotoxicity. Most of them act as effective inhibitors of the tumor-linked CA isoforms IX and XII, in nanomolar range. Also, different compounds have shown selectivity in comparable with the standard acetazolamide. Our IBS 5 d, 5 g, and 5 l (with Ki: 10.1, 19.4, 19.8 nM against hCA IX and 47, 45, 20 nM against hCA IX) showed the best inhibitory profile. In-vitro screening of all derivatives against a full sixty-cell-lined from NCI at a single dose of 10 µM offered growth inhibition of up to 45 %. Compound 5 b has been identified with the most potent cytotoxic activity and broad spectrum. Docking studies have also been implemented and were also in accordance with the biological outcomes. Our SAR analysis has interestingly proposed efficient tumor-related hCAs IX/XII suppression.

Hypoxia-related carbonic anhydrase 9 induces serpinB9 expression in cancer cells and apoptosis in T cells via acidosis.

Hypoxia is a common feature of solid tumors. However, the impact of hypoxia on immune cells within tumor environments remains underexplored. Carbonic anhydrase 9 (CA9) is a hypoxia-responsive tumor-associated enzyme. We previously noted that regardless of human CA9 (hCA9) expression, hCA9-expressing mouse renal cell carcinoma RENCA (RENCA/hCA9) presented as a "cold" tumor in syngeneic aged mice. This study delves into the mechanisms behind this observation. Gene microarray analyses showed that RENCA/hCA9 cells exhibited elevated mouse serpinB9, an inhibitor of granzyme B, relative to RENCA cells. Corroborating this, RENCA/hCA9 cells displayed heightened resistance to antigen-specific cytotoxic T cells compared with RENCA cells. Notably, siRNA-mediated serpinB9 knockdown reclaimed this sensitivity. In vivo tests showed that serpinB9 inhibitor administration slowed RENCA tumor growth, but this effect was reduced in RENCA/hCA9 tumors, even with adjunctive immune checkpoint blockade therapy. Further, inducing hypoxia or introducing the mouse CA9 gene upregulated serpinB9 expression, and siRNA-mediated knockdown of the mouse CA9 gene inhibited the hypoxia-induced induction of serpinB9 in the original RENCA cells. Supernatants from RENCA/hCA9 cultures had lower pH than those from RENCA, suggesting acidosis. This acidity enhanced serpinB9 expression and T cell apoptosis. Moreover, coculturing with RENCA/hCA9 cells more actively prompted T cell apoptosis than with RENCA cells. Collectively, these findings suggest hypoxia-associated CA9 not only boosts serpinB9 in cancer cells but also synergistically intensifies T cell apoptosis via acidosis, characterizing RENCA/hCA9 tumors as "cold."

Effective Anticancer Potential of a New Sulfonamide as a Carbonic Anhydrase IX Inhibitor Against Aggressive Tumors.

This study examines efficiency of a newly synthesized sulfonamide derivative 2-bromo-N-(4-sulfamoylphenyl)propanamide (MMH-1) on the inhibition of Carbonic Anhydrase IX (CA IX), which is overexpressed in many solid tumors including breast cancer. The inhibitory potential of MMH-1 compound against its four major isoforms, including cytosolic isoforms hCA I and II, as well as tumor-associated membrane-bound isoforms hCA IX and XII, was evaluated. To this context, the cytotoxic effect of MMH-1 on cancer and normal cells was tested and found to selectively affect MDA-MB-231 cells. MMH-1 reduced cell proliferation by holding cells in the G0/G1 phase (72 %) and slowed the cells' wound healing capacity. MMH-1 inhibited CA IX under both hypoxic and normoxic conditions and altered the morphology of triple negative breast cancer cells. In MDA-MB-231 cells, inhibition of CA IX was accompanied by a decrease in extracellular pH acidity (7.2), disruption of mitochondrial membrane integrity (80 %), an increase in reactive oxygen levels (25 %), and the triggering of apoptosis (40 %). In addition, the caspase cascade (CASP-3, -8, -9) was activated in MDA-MB-231 cells, triggering both the extrinsic and intrinsic apoptotic pathways. The expression of pro-apoptotic regulatory proteins (Bad, Bax, Bid, Bim, Cyt-c, Fas, FasL, TNF-a, TNF-R1, HTRA, SMAC, Casp-3, -8, P21, P27, and P53) was increased, while the expression of anti-apoptotic proteins, apoptosis inhibitor proteins (IAPs), and heat shock proteins (HSPs) (Bcl-2, Bcl-w, cIAP-2, HSP27, HSP60, HSP70, Survivin, Livin, and XIAP) was decreased. These results propose that the MMH-1 compound could triggers apoptosis in MDA-MB-231 cells via the pH/MMP/ROS pathway through the inhibition of CA IX. This compound is thought to have high potential and promising anticancer properties in the treatment of aggressive tumors.

Preclinical Evaluation of virus-like particle Vaccine Against Carbonic Anhydrase IX Efficacy in a Mouse Breast Cancer Model System.

Carbonic anhydrase IX (CAIX) is a cancer-associated membrane protein frequently overexpressed in hypoxic solid tumours leading to enhanced tumour cell survival and invasion, and it has been proposed to be an attractive tumour-specific molecule for antibody-mediated targeting. This study aimed to generate a virus-like particle (VLP)-based CAIX vaccine candidate and evaluate its efficacy in a mouse model of breast cancer. The prototype murine vaccine was developed based on the ssRNA bacteriophage Qbeta VLPs with chemically coupled murine CAIX protein catalytic domains on their surfaces. The vaccine was shown to efficiently break the natural B cell tolerance against autologous murine CAIX and to induce high-titre Th1-oriented IgG responses in the BALB/c mice. This vaccine was tested in a therapeutic setting by using a triple-negative breast cancer mouse model system comprising 4T1, 4T1-Car9KI and 4T1-Car9KO cells, the latter representing positive and negative controls for murine CAIX production, respectively. The humoural immune responses induced in tumour-bearing animals were predominantly of Th1-type and higher anti-mCAIXc titres correlated with slower growth and lung metastasis development of 4T1 tumours constitutively expressing mCAIX in vivo in the syngeneic host.

LncRNA ZNF674-AS1 drives cell growth and inhibits cisplatin-induced pyroptosis via up-regulating CA9 in neuroblastoma.

Neuroblastoma (NB) is a challenging pediatric extracranial solid tumor characterized by a poor prognosis and resistance to chemotherapy. Identifying targets to enhance chemotherapy sensitivity in NB is of utmost importance. Increasing evidence implicates long noncoding RNAs (lncRNAs) play important roles in cancer, but their functional roles remain largely unexplored. Here, we analyzed our RNA sequencing data and identified the upregulated lncRNA ZNF674-AS1 in chemotherapy non-responsive NB patients. Elevated ZNF674-AS1 expression is associated with poor prognosis and high-risk NB. Importantly, targeting ZNF674-AS1 expression in NB cells suppressed tumor growth in vivo. Further functional studies have revealed that ZNF674-AS1 constrains cisplatin sensitivity by suppressing pyroptosis and promoting cell proliferation. Moreover, ZNF674-AS1 primarily relies on CA9 to fulfill its functions on cisplatin resistance. High CA9 levels were associated with high-risk NB and predicted poor patient outcomes. Mechanistically, ZNF674-AS1 directly interacted with the RNA binding protein IGF2BP3 to enhance the stability of CA9 mRNA by binding with CA9 transcript, leading to elevated CA9 expression. As a novel regulator of CA9, IGF2BP3 positively upregulated CA9 expression. Together, these results expand our understanding of the cancer-associated function of lncRNAs, highlighting the ZNF674-AS1/IGF2BP3/CA9 axis as a constituting regulatory mode in NB tumor growth and cisplatin resistance. These insights reveal the pivotal role of ZNF674-AS1 inhibition in recovering cisplatin sensitivity, thus providing potential therapeutic targets for NB treatment.

Discovery of Novel Pyridazine-Tethered Sulfonamides as Carbonic Anhydrase II Inhibitors for the Management of Glaucoma.

As a progressive neuropathic condition, glaucoma can cause lifelong blindness if left untreated. Novel phenylpyridazine-tethered sulfonamides were designed as selective inhibitors for carbonic anhydrase (CA) isoform II to find effective therapeutic agents for glaucoma. Subsequently, the target inhibitors were synthesized and assessed for their inhibitory action against cytosolic CA I and II. Interestingly, the synthesized molecules poorly inhibited CA I while exhibiting low subnanomolar potency against CA II. Compound 7c disclosed the most potent activity (IC50 = 0.63 nM) with high selectivity against CA II (605-fold than acetazolamide selectivity). Moreover, compound 7c also showed significant in vivo IOP-reducing properties in the in vivo model of glaucoma. Furthermore, the binding of compound 7c to CA II was assessed at the molecular level, exploiting the molecular docking approach.

Exploring the potency of diazo-coumarin containing hybrid molecules: Selective inhibition of tumor-associated carbonic anhydrase isoforms IX and XII.

This study introduces a series of ten hybrid molecules DK(1-10), which combine diazo and coumarin moieties along with diverse aromatic substitutions. The primary objective was to evaluate the inhibitory capabilities of these compounds against four prominent isoforms: the cytosolic hCA I and II, as well as the tumor-associated membrane-bound hCA IX and XII. Impressively, the majority of the tested compounds exhibited significant inhibition activity against the tumor-associated isoforms hCA IX and XII, with KI values ranging from 29.2 to 293.3 nM. Notably, compound DK-8 displayed particularly robust inhibitory activity against the tumor-associated membrane-bound isoforms, hCA IX and XII, yielding KI values of 32.5 and 29.2 nM, respectively. Additionally, another derivative, DK-9, containing a primary sulfonamide, exhibited notable inhibition against hCA XII with a KI value of 36.4 nM. This investigation aimed to explore the structure-activity relationships within these compounds, shedding light on how various substitutions and structural components influence their inhibitory potential. As a result, these compounds present promising candidates for further exploration in medicinal and pharmacological research. Their ability to selectively inhibit specific isoforms, particularly those associated with hypoxic tumors, suggests their potential as foundational compounds for the development of novel therapeutic agents.

Identification of 3-(5-cyano-6-oxo-pyridin-2-yl)benzenesulfonamides as novel anticancer agents endowed with EGFR inhibitory activity.

New 5-cyano-6-oxo-pyridine-based sulfonamides (6a-m and 8a-d) were designed and synthesized to potentially inhibit both the epidermal growth factor receptor (EGFR) and carbonic anhydrase (CA), with anticancer properties. First, the in vitro anticancer activity of each target substance was tested using Henrietta Lacks cancer cell line and M.D. anderson metastasis breast cancer cell line cells. Then, the possible CA inhibition against the human CA isoforms I, II, and IX was investigated, together with the EGFR inhibitory activity, with the most powerful derivatives. The neighboring methoxy group may have had a steric effect on the target sulfonamides, which prevented them from effectively inhibiting the CA isoforms while effectively inhibiting the EGFR. The effects of the 5-cyanopyridine derivatives 6e and 6l on cell-cycle disruption and the apoptotic potential were then investigated. To investigate the binding mechanism and stability of the target molecules, thorough molecular modeling assessments, including docking and dynamic simulation, were performed.

Bifunctional drug delivery system with carbonic anhydrase IX targeting and glutathione-responsivity driven by host-guest amphiphiles for effective tumor therapy.

It remains a critical issue to deliver anticancer drugs to tumor tissues and reducing the toxic effects on normal tissues. The drug delivery system (DDS) based on self-assembly provides a multi-functional way for drug delivery. In this work, a supramolecular host (L-CD) with targeting function based on a ß-cyclodextrin (ß-CD) backbone was synthesized with carbonic anhydrase IX (CAIX) overexpressed on tumor cells as a target, and the methotrexate prodrug (MTX-SS-Ad) modified by adamantane and disulfide bond was prepared to be used as the guest. The amphiphilic complex was prepared between L-CD and MTX-SS-Ad through host-guest interactions and could further self-assemble into supramolecular nanoparticles (SNPs) with active targeting and stimulus release functions. The interaction between host and guest was investigated by UV, NMR, IR, XRD and TGA. The characteristic of SNPs was observed by DLS and TEM. Throng the study of molecular docking, in vitro inhibition, cell uptake experiments, and western blotting, SNPs have showed CAIX inhibitory effects both inside and outside the cells. The in vitro release experiments indicated that SNPs can undergo disintegration and release drugs under acidic and GSH conditions. Moreover, SNP can effectively inhibit the proliferation of cancer cells without generating additional toxic side effects on normal cells. So, we provide a strategy of bifunctional drug delivery system with targeting and glutathione-responsivity for effective tumor therapy.