Carbonic Anhydrase Isoforms Research Articles

Engineered protein-small molecule conjugates empower selective enzyme inhibition

Potent, specific ligands drive precision medicine and fundamental biology. Proteins, peptides, and small molecules constitute effective ligand classes. Yet greater molecular diversity would aid the pursuit of ligands to elicit precise biological activity against challenging targets. We demonstrate a platform to discover protein-small molecule (PriSM) hybrids to combine unique pharmacophore activities and shapes with constrained, efficiently engineerable proteins. In this platform, a fibronectin protein library is displayed on yeast with a single cysteine coupled to acetazolamide via a maleimide-poly(ethylene glycol) linker. Magnetic and flow cytometric sorts enrich specific binders to carbonic anhydrase isoforms. Isolated PriSMs exhibit potent, specific inhibition of carbonic anhydrase isoforms with efficacy superior to that of acetazolamide or protein alone, including an 80-fold specificity increase and 9-fold potency gain. PriSMs are engineered with multiple linker lengths, protein conjugation sites, and sequences against two different isoforms, which reveal platform flexibility and impacts of molecular designs. PriSMs advance the molecular diversity of efficiently engineerable ligands.

Transition‐Metal Complexes of Bidentate Schiff‐Base Ligands: In Vitro and In Silico Evaluation as Non‐Classical Carbonic Anhydrase and Potential Acetylcholinesterase Inhibitors

AbstractSchiff bases display superior features for many areas, such as significant intermediates in industrial biological, pharmacological, catalytic and optical properties, organic synthesis, and coordination chemistry. The pre‐synthesized two Schiff base ligands (HL1andHL2) and their bidentate metal complexes (Co(L1)2,Cu(L1)2,Ni(L1)2,Co(L2)2,Cu(L2)2, andNi(L2)2) were tested for their inhibition activities on acetylcholinesterase (AChE) and human carbonic anhydrase (hCA I andhCA II) isoforms. The transition metal complexes of bidentate Schiff base ligands displayed the potent inhibition effect withKIconstants ranging from 16.39±0.15 to 88.63±0.27 nM and 9.32±0.13 to 33.66±0.57 nM forhCA isoenzymes and AChE, respectively. The compoundCu(L1)2forhCA I andNi(L2)2for AChE andhCA II had the highest inhibitory effect. Besides, the molecular docking analyses of the most active complexes (Cu(L1)2andNi(L2)2) were performed to understand the binding interactions on the enzymes’ binding sites. According to both in vitro and in silico analysis results, all the compounds were potential inhibitors of AChE andhCA I, II isoenzymes.

Structure and mechanism of secondary sulfonamide binding to carbonic anhydrases.

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

Novel triazole-sulfonamide bearing pyrimidine moieties with carbonic anhydrase inhibitory action: Design, synthesis, computational and enzyme inhibition studies

A series of new triazole-sulfonamide bearing pyrimidine derivatives were designed and synthesized via click chemistry. All new compounds (SH-1 to SH-28) were validated by 1HNMR, 13CNMR, HRMS, and SH-3 was further structurally validated by X-Ray single diffraction study. These compounds (SH-1 to SH-28) were tested as inhibitors of human carbonic anhydrase (hCA) isoforms, such as hCA I, II, IX and XII, using a stopped flow CO2 hydrase assay. Most of the compounds exhibited significant inhibitory activity against hCA II and weak inhibitory activity against hCA I. The target compounds also displayed moderate to excellent inhibitory activity against tumor-related hCAs IX and XII. Some compounds, e.g., SH-20 (Ki = 9.4 nM), SH-26 (Ki = 1.8 nM) and SH-28 (Ki = 0.82 nM) exhibited excellent inhibitory activity and selectivity profile against hCAs XII over IX. SH-23 displayed promising inhibitory activity and selectivity profile against both tumor-related hCAs IX (Ki = 2.9 nM) as well as XII (Ki = 0.82 nM) over hCA I and II. To understand the molecular interactions, molecular docking study of compounds SH-20, SH-23, SH-26 and SH-28 with hCA XII and SH-23 also with hCA IX were performed. The computational study evidenced favorable interaction between the inhibitors and active residues of both proteins. Some of these derivatives are promising leads for the development of selective, anticancer agents based on CA inhibitors.

Quantum mechanical study on the activation mechanism of human carbonic anhydrase VII cluster model with bis-histamine schiff bases and bis-spinaceamine derivatives

The activation mechanism of human carbonic anhydrase (hCA) isoform VII, hCA VII, with histamine, histamine bis-Schiff bases and bis-spinaceamine derivatives has been investigated using quantum mechanical calculations. The DFT-D3 method has been employed to calculate in detail the electronic structure and electronic energy of different compounds and complexes throughout the reaction pathway. The model system of hCA VII included the core catalytic center, the Zn2+ ion, its three histidine ligands and a hydroxide ion or water molecule coordinated to it. Furthermore, Thr199, Glu106 and the deep water molecule were considered in the model. Five activators of this enzyme, including histamine as standard, in complex with the cluster model of hCA VII were investigated. Thermodynamic functions for the overall reaction and for the complexation between activators and hCA VII were evaluated. Our results demonstrate that the protonatable moiety of these activators participates in proton transfer reactions from the zinc-bound water molecule to the reaction medium, promoting the formation of the catalytically active zinc hydroxide species of the enzyme. The QM analysis revealed that the electrostatic interactions between activators and hCA VII are the driving force of the enzyme-activator complex formation.

Inhibition of Carbonic Anhydrase-IX As a Novel Strategy to Overcome Hypoxia Mediated Drug Resistance in FLT3/ITD Mutated Acute Myeloid Leukemia

Background: FLT3/ITD mutated (FLT3/ITD+) acute myeloid leukemia (AML) is an aggressive form of blood cancer that carries a poor prognosis due to short remission durations and high relapse rates. Several studies suggest that this poor outcome is due to a small fraction of leukemic cells (LCs) that elude treatment and survive in hypoxic niches of the bone marrow (BM) where they lead to relapse. The identification of molecules capable to target LCs under hypoxic conditions is therefore of utmost importance on the way to find a cure for AML. Our study addresses the effects of pharmacological inhibition of carbonic anhydrase-IX (CA9), a transmembrane protein which functions to maintain a neutral intracellular pH - a key survival mechanism for tumor cells to escape cell death in the hypoxic environment - on AML cells. Targeting CA9 is of particular interest as its expression is confined to only a few normal tissues. CA9 inhibition may therefore show relatively few side effects compared to standard chemotherapies. Methods: Molm14 (M14), M13, OCI-AML3, THP-1 and primary cells (PC) from FLT3/ITD+ AML patients were incubated under normoxic (21% O2) and hypoxic (1% O2) conditions in the presence or absence of Quizartinib (Quiz), Cytarabine (Cy) or the CA9 inhibitors FC531 (FC) or SLC-0111 (SLC). After 48h proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis. Cells were also assessed for CA9 and CA12 mRNA expression as well as intracellular pH levels via RT-PCR and fluorescent imaging, respectively. FLT3/ITD+ AML xenografts were generated by injecting M14 cells in the tail vein of NSG mice. Tissue samples were fixed in 10% NBF, embedded in paraffin and stained with H&E and CA9 for pathologic evaluation. Results: CA9 mRNA was upregulated in M14 cells (3.8±1.1-fold; n=3, p<.05) and FLT3/ITD+ AML PCs (215.4±142.7-fold, n=7, p=NS) under 1% compared to 21% O2. Consistently, immunohistochemical staining of M14 xenograft tissues showed multifocal CA9 staining of LCs in the BM and spleen. In M14 cells, inhibition of CA9 with FC conferred substantial, dose-dependent growth inhibitory effects under 1% O2 (IC50=4.04µM) and was significantly more effective than clinically achievable doses of Quiz or Cy in this regard. Under 1% O2, flow cytometry revealed significant induction of apoptosis in FC, but not in Quiz or Cy treated cells (p<.01 vs Cy, p<.01 vs Quiz; n=3). Similar results were obtained in M13 cells. Treatment of M14 cells with 5µM FC under 1% O2 resulted in substantial intracellular pH acidification (ΔpH=-2.7±0.6, p<.05 vs 1% O2, n=3) within 48h. FC synergistically inhibited M14 cell growth when combined with Cy (CI=0.78). To investigate the effects of CA9 inhibition against non-FLT3 mutated cells we incubated OCI-AML3 and THP-1 cells to increasing doses of FC (1-10µM) under 1% O2. We observed that both cell lines were less sensitive to FC (IC50>10µM) compared to M14 and M13 cells. Interestingly, these cell lines displayed higher levels of CA12 mRNA (CA9/12 ratio=0.68 [OCI-AML3] and 0.54 [THP-1]) compared to the FC sensitive cell lines M14 and M13 (CA-9/12 ratio=2.5 and 4.6, respectively). Next, we assessed the effects Cy (1µM), Quiz (100nM) and FC (200µM) against FLT/ITD+ PCs. As expected, Cy and Quiz did not confer cytotoxic effects under 1% O2. Surprisingly, FC was only mildly effective against FLT/ITD+ PCs, which harbored low CA9/12 ratios (<0.5). In contrast, treatment with equimolar doses of SLC, a different class CA9 inhibitor, exhibited substantial anti-leukemic activity under 1% O2 conditions (78.8±1.9% growth inhibition, n=3, p<.05 vs FC, p<.001 vs Cy or Quiz). Conclusions: 1. CA9 is upregulated in FLT3/ITD+ cell lines and PCs under hypoxic conditions. 2. CA9 is expressed in LCs residing in the BM and spleen of FLT/ITD+ AML xenografts. 3. The CA9 inhibitor FC531 confers marked anti-leukemic activity against FLT3/ITD+ AML cells under hypoxic conditions via intracellular pH acidification and acts synergistically with cytarabine. 4. Upregulation of other carbonic anhydrase isoforms, such as CA12, might represent a resistance mechanism against FC531 that can be overcome by different class CA9 inhibitors. 5. Our data suggests that CA9 inhibition has the potential to specifically target LCs residing in hypoxic niches and may be of value as an adjunct to chemotherapy for FLT3/ITD+AML treatment. 6. Our ongoing work evaluates the effects of CA9 inhibition in AML xenografts. DisclosuresNo relevant conflicts of interest to declare.

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

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

Efficacy of a Three Drug-Based Therapy for Neuroblastoma in Mice.

High-risk neuroblastoma (HR-NB) still remains the most dangerous tumor in early childhood. For this reason, the identification of new therapeutic approaches is of fundamental importance. Recently, we combined the conventional pharmacological approach to NB, represented by cisplatin, with fendiline hydrochloride, an inhibitor of several transporters involved in multidrug resistance of cancer cells, which demonstrated an enhancement of the ability of cisplatin to induce apoptosis. In this work, we co-administrated acetazolamide, a carbonic anhydrase isoform IX (CAIX) inhibitor which was reported to increase chemotherapy efficacy in various cancer types, to the cisplatin/fendiline approach in SKNBE2 xenografts in NOD-SCID mice with the aim of identifying a novel and more effective treatment. We observed that the combination of the three drugs increases more than twelvefold the differences in the cytotoxic activity of cisplatin alone, leading to a remarkable decrease of the expression of malignancy markers. Our conclusion is that this approach, based on three FDA-approved drugs, may constitute an appropriate improvement of the pharmacological approach to HR-NB.

Synthesis, biological activity and docking calculations of bis-naphthoquinone derivatives from Lawsone

Some metabolic enzyme inhibitors can be used as Multi-target-Directed-Ligands (MTDL) in Medicinal chemistry therefore, synthesis and determination of alternative inhibitors are essential. In this study, novel bis-napthoquinone derivatives (5a-o) were synthesized through a multi-component cascade reaction of two molecules of 2-hydroxy-1,4-naphthoquinone with an aromatic aldehyde in basic media using triethylamine as a catalyst. This novel heterocyclic derivatives (5a-o) are applied to inhibit the carbonic anhydrase (hCA I and hCA II) isoform in low levels of nano molecules with Ki values exist between 4.62 ± 1.01 to 70.45 ± 9.03 nM for hCA I and for hCA II which is physiologically dominant Kis values are in the range of 5.61 ± 1.04 to 73.26 ± 10.25 nM. Further these novel derivatives (5a-o) efficiently inhibit AChE with Ki values in the range of 0.13 ± 0.02 to 3.16 ± 0.56 nM. The compounds are also applied for BChE with Ki values varying between 0.50 ± 0.10 to 9.23 ± 1.15 nM. For α-glycosidase, the most efficient Ki values of 5e and 5f are 76.14 ± 9.60 and 95.27 ± 12.55 nM respectively. Finally, molecular docking calculations against enzymes (acetylcholinesterase, butyrylcholinesterase, and the human carbonic anhydrase I and II) are compared using biological activities of heterocyclic derivatives. After these calculations, an ADME/T analysis is performed to study the future medicinal use of heterocyclic derivatives from lawsone.

Potential anticancer activities of Rhus coriaria (sumac) extract against human cancer cell lines.

Therapeutic strategies of plant origin are a better choice as both dietary plant products or its isolated active constituents against the development and progression of cancer. The present study aims to evaluate the anticancer activity of sumac (Rhus coriaria) against different human cancer MCF-7, PC-3, and SKOV3 cell lines. In addition, the study tries to explore a prospective mechanism of action, assessment of in vitro enzyme-inhibitory capacity of sumac extract against hCA I, II, IX, and XII. In the present study, the potential antitumor effects of sumac (Rhus coriaria) were explored in the human cancer cell lines; MCF-7, PC-3, and SKOV3 using in vitro assays. Apoptotic, cell survival, ELISA immunoassays were also conducted to reveal the inhibitory effects of sumac extract against hCA I, II, IX, and XII. In addition, both Clioquinol and Acetazolamide (AZM) were used as standards to explore the in vitro enzyme-inhibitory capacity of sumac extract against hCA I, II, IX, and XII. The hydro-alcoholic extract of R. coriaria (Sumac) was subjected to phytochemical analysis using GC/MS assays. Sumac at non-cytotoxic doses of 50 and 100 µM significantly modulates the growth of the MCF-7, PC-3, and SKOV3 cancer cells with a higher inhibitory effect and selectivity to carbonic anhydrase (CA) isoforms; hCA I, II, hCA IX, and XII. The data showed that sumac at doses of 50 and 100 µM significantly inhibited the growth, proliferation, and viability of cancer cells by activating the apoptotic process via caspase-3 overexpression and the regulation of Bcl-2 anti-apoptotic protein.

Comparative Proteomics Analysis of the Post‐Mitochondrial Fraction (S9 Fraction) of Human Lens‐Free Whole Eye and Liver

Background The increasing incidence of ocular diseases has accelerated research into therapeutic interventions for the eye. The knowledge of key metabolic mechanisms in the eye is vital for the successful ophthalmic drug discovery. The lens-free whole-eye post-mitochondrial fraction(S9 fraction) is one of the most widely used in vitro system for ocular metabolism studies during early drug discovery stage. As of now, no report of a comprehensive proteomics investigation exists for the pooled ocular S9 fraction. The objective of this study was to explore the human ocular S9 proteome and compare it with the human liver S9 proteome. Methods The global proteomics study was performed on the pooled ocular (n=11) and hepatic (n=50) S9 fraction samples in data-dependent acquisition mode using an optimized protocol. The samples were acquired in triplicate to minimize the technical variability. The acquired proteomics data were analyzed using Maxquant with 1% false discovery rate. A FASTA file containing whole human proteome, was used as the reference sequence for the search in the Andromeda database search tool. Additionally, another FASTA file containing 1187 proteins involved in the metabolism of endobiotics and xenobiotics was also searched to specifically identify the metabolic enzymes in both organs. The proteins were quantified using a generic label free quantification method (MaxLFQ) in Maxquant software. The metabolic enzymes common to both eye and liver S9 fractions were compared to determine the relative abundance values based on the resulting MaxLFQ intensities. The cut off for the differentially expressed proteins between the eye or liver was either <0.5 or >2. Results and Conclusion A total of 269 proteins (including 23 metabolic enzymes) were detected exclusively in the pooled eye S9, against 648 proteins in the liver S9 (including 174 metabolic enzymes), whereas 424 proteins (including 94 metabolic enzymes) were detected in both the organs. Among the common metabolic enzymes, 18 were predominant in the eye (Figure 1), 43 were predominant in the liver, while rest 33 were comparable in both samples. The major DMEs, viz., Cytochrome P450s, UDP-glucuronosyltransferases, and sulfotransferases, were not detected in the eye S9 fraction, instead isoforms of alcohol dehydrogenases, aldehyde dehydrogenases, carbonic anhydrases, aldo-keto reductases, esterases, epoxide hydrolases, and glutathione S-transferases were in abundance. This information bears great significance in gaining deeper insights on metabolic and bioactivation potential of both systemically as well as topically administered ophthalmic drugs as well as in the endobiotics metabolism within the eye. This investigation provides foundation for the subsequent targeted proteomic, genomic, and functional studies to explore the effects of sex, age, genotype and disease conditions across the populations.

Switching the Inhibitor‐Enzyme Recognition Profile via Chimeric Carbonic Anhydrase XII

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.

Inhibition studies on carbonic anhydrase isoforms I, II, IV and IX with N-arylsubstituted secondary sulfonamides featuring a bicyclic tetrahydroindazole scaffold

Carbonic Anhydrases (CAs) are pharmaceutically relevant targets for the treatment of several disease conditions. The ubiquitous localization of these enzymes and the high homology shared by the different isoforms represent substantial impediments for the discovery of potential drugs devoid of off-target side effects. As a consequence, substantial efforts are still needed to allow for the full realization of the pharmacological potential of CA modulators. In this contribution, starting from our previous studies, we describe the synthesis of a set of new bicyclic tetrahydroindazoles featuring a secondary sulfonamide. Biological evaluation of the inhibitory activity against the hCA I, II, IV, and IX isoforms allowed drawing a structure-activity relationship profile that was rationalized through theoretical studies. This allowed dissecting the new molecules into the single portions influencing the zinc chelation properties and the selectivity profile thereby offering a new platform for the discovery of new isotype selective CA inhibitors.

Developments of small molecules as inhibitors for carbonic anhydrase isoforms

Carbonic anhydrases are ubiquitous, and their role in the hydration of carbon dioxide is essential for the survival of many tissues and organs. However, their association with many pathological diseases, especially in glaucoma, Alzheimer's, obesity, epilepsy, and tumorigenesis, has prompted the design and synthesis of novel carbonic anhydrase inhibitors (CAIs). Herein we describe (1) approaches used in the design of CAIs and (2) synthesis of small molecules as CAIs within the last five years. Despite the active research in this area, there are still more avenues to explore, especially selective inhibition of CA I, CA IX, and XII. These isoforms would continue to open up a diversity of carbonic anhydrase inhibitors containing 1,2,3-triazoles, imidazolone, pyrrolidone, thiadiazole, isatin, and glycoconjugates as part of their molecular frameworks.

Synthesis and Biological Evaluation of Coumarin-Linked 4-Anilinomethyl-1,2,3-Triazoles as Potent Inhibitors of Carbonic Anhydrases IX and XIII Involved in Tumorigenesis.

A series of coumarin-linked 4-anilinomethyl-1,2,3-triazoles (6a–t) was synthesized via a molecular hybridization approach, through carbon C-6 of the coumarin moiety. The synthesized compounds were evaluated for their inhibition of carbonic anhydrase (CA) isoforms I, II, IX and XIII. CAs IX and XIII were selectively inhibited over the off-target isoforms I and II. The best inhibitory profiles against CA IX were shown by compounds 6a, 6e and 6f (Ki < 50 nM), with compound 6e displaying the best inhibition with a Ki value of 36.3 nM. Compounds 6a, 6b, 6j, 6o and 6q exhibited the best inhibitory profiles against CA XIII (Ki < 100 nM). These compounds can be further explored for the discovery of potent and effective CA IX and CA XIII inhibitors.

Effect of an Imidazole-Containing Schiff Base of an Aromatic Sulfonamide on the Cytotoxic Efficacy of N,N-Coordinated Half-Sandwich Ruthenium(II) p-Cymene Complexes

Sulfonamides have a broad range of therapeutic applications, which include the inhibition of various isoforms of carbonic anhydrases (CAs). Among the various CA isoforms, CA IX is overexpressed in tumors and regulates the pH of the tumor microenvironment. Herein we present five new ruthenium(II) p-cymene complexes (1-5) of Schiff base ligands (L1-L4) of 4-(2-aminoethyl)benzenesulfonamide by varying the aldehyde to enhance the selective cytotoxicity toward cancer cells. All of the complexes are stable to aquation for the observed period of 24 h except 1, which aquated within 1 h, but the monoaquated species is stable for 24 h. The two imidazole derivatives, 1 and 2, are cytotoxic to the cancer cells MDA-MB-231 and MIA PaCa-2 but not to the noncancerous cells CHO and MDCK. The enhanced toxicity in hypoxia against MDA-MB-231 may be due to the greater expression of CA IX in hypoxia, as per the immunofluorescence data. The most cytotoxic complexes, 1 and 2, are lipophilic, whereas 3-5 show high hydrophilicity and are not cytotoxic up to 200 μM. Complexes 1 and 2 also show a higher cellular accumulation in MDA-MB-231 than the nontoxic yet solution-stable complex 5. The cytotoxic complexes bind with the model nucleobase 9-ethylguanine but have slow reactivity toward cellular tripeptide glutathione. Both 1 and 2 induce apoptosis by depolarizing the mitochondrial membrane potential and arrest the cell cycle in the SubG1 phase.

Proton Transport in Cancer Cells: The Role of Carbonic Anhydrases.

Intra- and extracellular pH regulation is a pivotal function of all cells and tissues. Net outward transport of H+ is a prerequisite for normal physiological function, since a number of intracellular processes, such as metabolism and energy supply, produce acid. In tumor tissues, distorted pH regulation results in extracellular acidification and the formation of a hostile environment in which cancer cells can outcompete healthy local host cells. Cancer cells employ a variety of H+/HCO3−-coupled transporters in combination with intra- and extracellular carbonic anhydrase (CA) isoforms, to alter intra- and extracellular pH to values that promote tumor progression. Many of the transporters could closely associate to CAs, to form a protein complex coined “transport metabolon”. While transport metabolons built with HCO3−-coupled transporters require CA catalytic activity, transport metabolons with monocarboxylate transporters (MCTs) operate independently from CA catalytic function. In this article, we assess some of the processes and functions of CAs for tumor pH regulation and discuss the role of intra- and extracellular pH regulation for cancer pathogenesis and therapeutic intervention.

Insertion of metal carbenes into the anilinic N-H bond of unprotected aminobenzenesulfonamides delivers low nanomolar inhibitors of human carbonic anhydrase IX and XII isoforms.

Herein we report the synthesis of a set of thirty-four primary sulfonamides generated via formal N-H-insertion of metal carbenes into anilinic amino group of sulfanilamide and its meta-substituted analog. Obtained compounds were tested invitro as inhibitors of four physiologically significant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Many of the synthesized sulfonamides displayed low nanomolar Ki values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Provided the promising activity profiles of the substances towards tumor-associated hCA IX and XII isozymes, single-concentration MTT test was performed for the entire set. Disappointingly, most of the discovered hCA inhibitors did not significantly suppress the growth of cancer cells either in normoxia or CoCl2 induced hypoxic conditions. The only two compounds exerting profound antiproliferative effect turned out to be modest hCA inhibitors. Their out of the range activity in cells is likely attributive to the presence of Michael acceptor substructure which can potentially act either through the inhibition of Thioredoxin reductases (TrxRs, EC 1.8.1.9) or nonspecific covalent binding to cell proteins.

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2, intracellular carbonic anhydrase (CAi) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2-(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.

Prediction of activity and selectivity profiles of human Carbonic Anhydrase inhibitors using machine learning classification models

The development of selective inhibitors of the clinically relevant human Carbonic Anhydrase (hCA) isoforms IX and XII has become a major topic in drug research, due to their deregulation in several types of cancer. Indeed, the selective inhibition of these two isoforms, especially with respect to the homeostatic isoform II, holds great promise to develop anticancer drugs with limited side effects. Therefore, the development of in silico models able to predict the activity and selectivity against the desired isoform(s) is of central interest. In this work, we have developed a series of machine learning classification models, trained on high confidence data extracted from ChEMBL, able to predict the activity and selectivity profiles of ligands for human Carbonic Anhydrase isoforms II, IX and XII. The training datasets were built with a procedure that made use of flexible bioactivity thresholds to obtain well-balanced active and inactive classes. We used multiple algorithms and sampling sizes to finally select activity models able to classify active or inactive molecules with excellent performances. Remarkably, the results herein reported turned out to be better than those obtained by models built with the classic approach of selecting an a priori activity threshold. The sequential application of such validated models enables virtual screening to be performed in a fast and more reliable way to predict the activity and selectivity profiles against the investigated isoforms.