The development of a brief screener for autism using item response theory.

BACKGROUND: Brief screening instruments focusing on autism spectrum disorder (ASD) that can be administered in primary care are scarce; there is a need for shorter and more precise instruments. The Autism-Tics, AD/HD and other Comorbidities inventory (A-TAC) has previously been validated for ASD reporting excellent validity. This study aims to determine the psychometric properties of each item in the ASD domain (17 items) in the A-TAC using item response theory (IRT), and thereby construct and validate a short form that could be used as a screening instrument in the general population. METHODS: Since 2004, parents of all 9-year-old Swedish twins have been invited to participate in a telephone interview in the Child and Adolescent Twin Study in Sweden (CATSS). The CATSS is linked to the National Patient Register (NPR), which includes data from in- and outpatient care. Data on ASD (A-TAC) collected in CATSS were compared with diagnoses from the NPR. Diagnoses that had been made both before (previous validity) and after (predictive validity) the interviews were included. The sample was divided into a developmental sample and a validation sample. An IRT model was fitted to the developmental sample and item parameters were used to select a subset of items for the short form. The performance of the proposed short form was examined in the validation sample by the use of receiver operation characteristic curves. RESULTS: Four items which were able to discriminate among individuals with more autism traits were deemed sufficient for use in the short form. The values of the area under the receiver operating characteristic curve for a clinical diagnosis of ASD was .95 (previous validity) and .72 (predictive validity). CONCLUSIONS: The proposed short form with 4 out of the original 17 items from A-TAC, showed excellent previous validity while the predictive validity was fair. The validity of the short form was in agreement with previous validations of the full ASD domain. The short form can be a valuable screening instrument in primary care settings in order to identify individuals in need for further assessment and for use in epidemiological studies.

Plasmatic carbonic anhydrase IX as a diagnostic marker for clear cell renal cell carcinoma.

Carbonic anhydrase (CA, EC 4.2.1.1) IX is regarded as a tumour hypoxia marker and CA inhibitors have been proposed as a new class of antitumor agents, with one such agent in Phase II clinical trials. The expression of some CAs, in particular the isoforms CA IX and CA XII, has been correlated with tumour aggressiveness and progression in several cancers. The aim of this study was to evaluate the possibility that CA IX could represent a marker related to clear cell Renal Cell Carcinoma (ccRCC). Bcl-2 and Bax, and the activity of caspase-3, evaluated in tissue biopsies from patients, were congruent with resistance to apoptosis in ccRCCs with respect to healthy controls, respectively. In the same samples, the CA IX and pro-angiogenic factor VEGF expressions revealed that both these hypoxia responsive proteins were strongly increased in ccRCC with respect to controls. CA IX plasma concentration and CA activity were assessed in healthy volunteers and patients with benign kidney tumours and ccRCCs. CA IX expression levels were found strongly increased only in plasma from ccRCC subjects, whereas, CA activity was found similarly increased both in plasma from ccRCC and benign tumour patients, compared to healthy volunteers. These results show that the plasmatic level of CA IX, but not the CA total activity, can be considered a diagnostic marker of ccRCCs. Furthermore, as many reports exist relating CA IX inhibition to a better outcome to anticancer therapy in ccRCC, plasma levels of CA IX could be also predictive for response to therapy.

Development and biological evaluation of 99mTc-sulfonamide derivatives for in vivo visualization of CA IX as surrogate tumor hypoxia markers.

In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. In this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, K(i) = 33 nM for CA IX) has been conjugated with bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG2) tetradendate ligands and the conjugates radiolabelled with (99m)Tc, to obtain anionic and neutral (99m)Tc-labeled sulfonamide derivatives, respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory activities against hCA IX (K(i) = 59-66 nM). In addition, a second generation bis AEBS was conjugated with MAG2 and labeled with (99m)Tc, and the obtained diastereomers were also evaluated in targeting CA IX. Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG2 tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28% intact). This preliminary data suggest that negatively charged (99m)Tc-labeled sulfonamide derivatives with modest lipophilicity and longer circulation time could be promising markers to target CA IX.

Structural modulation of the biological activity of gold nanoparticles functionalized with a carbonic anhydrase inhibitor.

Gold nanoparticles (AuNPs) have gained attention for their potential and application in different fields, e.g. nanomedicine. This study explores the surface functionalization of AuNP with inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1). Some CA transmembrane isoforms have been recognized as therapeutic targets for the treatment of hypoxic tumors. Embedding a CA inhibitory function onto a nanosized unit has been proved to enable selective targeting of transmembrane isoforms. We report the preparation in aqueous media, the characterization and CA inhibition tests of AuNPs coated with a sulfonamide (SA) derivative, already known for its inhibitory activity toward CAs. The physico-chemical characterization of SA-coated AuNPs was performed with a combination of scattering and spectroscopic techniques. We detect a threshold effect of the SA concentration on the final hydrodynamic and core sizes of the capped nanoparticles and on their stability over aggregation. These modified nanoparticles were assayed for inhibition of some CA transmembrane isoforms (CA IX and XII) as well as of two cytosolic isoforms (CA I and II), and show interesting inhibitory efficiency in the submicromolar range and some selectivity for transmembrane isoforms.

Carbamoylphosphonates control tumor cell proliferation and dissemination by simultaneously inhibiting carbonic anhydrase IX and matrix metalloproteinase-2. Toward nontoxic chemotherapy targeting tumor microenvironment.

Carbamoylphosphonates (CPOs) have been identified as inhibitors of matrix metalloproteinases (MMPs) and as orally active, bioavailable, and safe antimetastatic agents. In this article, we focus on the direct antitumor activity of the CPOs. We discovered that CPOs also inhibit carbonic anhydrases (CAs), especially the IX and XII isoforms identified as cancer promoting factors. Thus, CPOs can be regarded as novel nontoxic drug candidates for tumor microenvironment targeted chemotherapy acting by two synergistic mechanisms, namely, inhibiting CAs and MMPs simultaneously. We have also demonstrated that the ionized CPO acid is unable to cross the cell membrane and thus limited to interact with the extracellular domains of isozymes CAIX and CAXII. Finally, applying CPOs against cancer cells in hypoxic conditions resulted in the dose dependent release of lactate dehydrogenase, confirming the direct interaction of the CPOs with the cancer related isozymes CAIX and XII and thereby promoting cellular damage.

Rational drug repositioning guided by an integrated pharmacological network of protein, disease and drug.

BACKGROUND: The process of drug discovery and development is time-consuming and costly, and the probability of success is low. Therefore, there is rising interest in repositioning existing drugs for new medical indications. When successful, this process reduces the risk of failure and costs associated with de novo drug development. However, in many cases, new indications of existing drugs have been found serendipitously. Thus there is a clear need for establishment of rational methods for drug repositioning. RESULTS: In this study, we have established a database we call "PharmDB" which integrates data associated with disease indications, drug development, and associated proteins, and known interactions extracted from various established databases. To explore linkages of known drugs to diseases of interest from within PharmDB, we designed the Shared Neighborhood Scoring (SNS) algorithm. And to facilitate exploration of tripartite (Drug-Protein-Disease) network, we developed a graphical data visualization software program called phExplorer, which allows us to browse PharmDB data in an interactive and dynamic manner. We validated this knowledge-based tool kit, by identifying a potential application of a hypertension drug, benzthiazide (TBZT), to induce lung cancer cell death. CONCLUSIONS: By combining PharmDB, an integrated tripartite database, with Shared Neighborhood Scoring (SNS) algorithm, we developed a knowledge platform to rationally identify new indications for known FDA approved drugs, which can be customized to specific projects using manual curation. The data in PharmDB is open access and can be easily explored with phExplorer and accessed via BioMart web service (http://www.i-pharm.org/, http://biomart.i-pharm.org/).

[(Cp-R)M(CO)3] (M=Re or 99mTc) Arylsulfonamide, arylsulfamide, and arylsulfamate conjugates for selective targeting of human carbonic anhydrase IX.

Enhanced receptor selectivity: carbonic anhydrase inhibitors are relevant for both cancer diagnosis and therapy. Combining non-radioactive Re compounds with their radioactive (99m)Tc homologs enables the use of identical molecules for therapy and imaging (theragnostic). The syntheses and in vitro evaluation of [(Cp-R)M(CO)(3)] (Cp=cyclopentadienyl, M=Re, (99m)Tc) with R being a highly potent carbonic-anhydrase-targeting vector is reported.

Carbonic anhydrase VII is S-glutathionylated without loss of catalytic activity and affinity for sulfonamide inhibitors.

Human carbonic anhydrase (CA, EC 4.2.1.1) VII is a cytosolic enzyme with high carbon dioxide hydration activity. Here we report an unexpected S-glutathionylation of hCA VII which has also been observed earlier in vivo for hCA III, another cytosolic isoform. Cys183 and Cys217 were found to be the residues involved in reaction with glutathione for hCA VII. The two reactive cysteines were then mutated and the corresponding variant (C183S/C217S) expressed. The native enzyme, the variant and the S-glutathionylated adduct (sgCA VII) as well as hCA III were fully characterized for their CO(2) hydration, esterase/phosphatase activities, and inhibition with sulfonamides. Our findings suggest that hCA VII could use the in vivo S-glutathionylation to function as an oxygen radical scavenger for protecting cells from oxidative damage, as the activity and affinity for inhibitors of the modified enzyme are similar to those of the wild type.

Synthesis of rhodamine B-benzenesulfonamide conjugates and their inhibitory activity against human α- and bacterial/fungal ß-carbonic anhydrases.

A series of fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors were obtained by attaching rhodamine B moieties to the scaffold of benzenesulfonamides. The new compounds have been investigated for the inhibition of 12 human α-CA isoforms (hCA I-hCA XIV), three bacterial and one fungal ß-class enzymes from the pathogens Mycobacterium tuberculosis and Candida albicans. All types of inhibitory activities have been detected, with several compounds showing low nanomolar inhibition against the transmembrane isoforms hCA IX, XII (cancer-associated) and XIV. The ß-CAs were inhibited in the micromolar range by these compounds which may have applications for the imaging of hypoxic tumors or bacteria due to their fluorescent moieties.

Carbonic anhydrase inhibitors. Synthesis, molecular structures, and inhibition of the human cytosolic isozymes I and II and transmembrane isozymes IX, XII (cancer-associated) and XIV with novel 3-pyridinesulfonamide derivatives.

A series of novel 3-pyridinesulfonamide derivatives (2-5, 9-11 and 13-15) have been synthesized and investigated as inhibitors of five isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is, the cytosolic ubiquitous CA I and II, and isozymes CA IX and XII (cancer-associated), and XIV. Against the human isozyme hCA I the new compounds showed K(I)s in the range of 0.089-251 µM, whereas toward hCA II, K(I)s = 50.5-487 nM. Isozyme hCA IX was inhibited with K(I)s in the range of 5.2-18.3 nM, while hCA XII with K(I)s = 6.0-16.4 nM, and hCA XIV with K(I)s = 76.4-152.0 nM. All of the new compounds 2-5, 9-11 and 13-15 showed excellent hCA IX inhibitory efficacy, with K(I)s = 5.2-18.3 nM, being much more effective as compared to the clinically used AAZ, MZA, EZA, DCP and IND (K(I)s = 24-50 nM).

Phenylethynylbenzenesulfonamide regioisomers strongly and selectively inhibit the transmembrane, tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic isoforms I and II.

A series of compounds incorporating regioisomeric phenylethynylbenzenesulfonamide moieties has been investigated for the inhibition of four human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms, hCA I, II, IX and XII. Inhibition between the low nanomolar to the milliomolar range has been observed against them, with several low nanomolar and tumor-CA selective inhibitors detected. The position of the sulfamoyl group with respect to the alkyne functionality, and the nature of the moieties substituting the second aromatic ring were the principal structural features influencing CA inhibition. The para-sulfamoyl-substituted derivatives were effective inhibitors of CA IX and XII, the meta-substituted regioisomers of CA I, IX and XII, whereas the ortho-substituted sulfonamides were weak inhibitors of CA I, II and IX, but inhibited significantly CA XII.

Kinetic and docking studies of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX and XII evidence a new binding mode within the enzyme active site.

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, coumarins (acting as prodrugs) and polyamines. A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) in a different manner, is reported here. Kinetic measurements allowed us to identify hydroxy-/methoxy-substituted benzoic acids as well as di-/tri-methoxy benzenes as submicromolar-low micromolar inhibitors of the four CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II allowed us to understand the inhibition mechanism. This new class of inhibitors binds differently compared to all other classes of inhibitors known to date: they were found between the phenol-binding site and the coumarin-binding site, filling thus the middle of the enzyme cavity. They exploit different interactions with amino acid residues and water molecules from the CA active site compared to other classes of inhibitors, offering the possibility to design CAIs with an interesting inhibition profile compared to the clinically used sulfonamides/sulfamates.

Design, synthesis, and biological evaluation of novel carbohydrate-based sulfamates as carbonic anhydrase inhibitors.

Carbonic anhydrases (CAs) IX and XII are enzymes with newly validated potential for the development of personalized, first-in-class cancer chemotherapies. Here we present the design and synthesis of novel carbohydrate-based CA inhibitors, several of which were very efficient inhibitors (K(i)<10 nM) with good selectivity for cancer-associated CA isozymes over off-target CA isozymes. All inhibitors comprised a carbohydrate core with one hydroxyl group derivatized as a sulfamate. Five different carbohydrates were chosen to present a selection of molecular shapes with subtle stereochemical differences to the CA enzymes active site. Variable modifications of the remaining sugar hydroxyl groups were incorporated to provide an incremental coverage of chemical property parameters that are associated with biopharmaceutical performance. All sulfamate inhibitors displayed ligand efficiencies that are consistent with those reported for good drug lead candidates.

Carbonic anhydrase inhibitors. Antioxidant polyphenols effectively inhibit mammalian isoforms I-XV.

A series of polyphenolic derivatives, including resveratrol, dobutamine, curcumin, catechin and silymarine were investigated for the inhibition of all the catalytically active mammalian isozymes of the metalloprotein carbonic anhydrase (CA, EC 4.2.1.1), that is, CA I-CA XV. These polyphenols effectively inhibited CAs, with K(I)s in the range of 380 nM-12.02 microM. The various isozymes showed quite diverse inhibition profiles with these compounds, which possess scaffolds not present in other investigated CA inhibitors (CAIs). These data may lead to drug design campaigns of effective CAIs possessing a diverse inhibition mechanism compared to sulfonamide/sulfamate inhibitors, based on such less investigated scaffolds.

Inhibition of the R1 fragment of the cadmium-containing zeta-class carbonic anhydrase from the diatom Thalassiosira weissflogii with anions.

We investigated the catalytic activity and inhibition of both the zinc and cadmium-containing R1 fragment of the zeta-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii. Our data prove that these enzymes are not only very efficient catalysts for the physiological reaction, but also sensitive to sulfonamide and anion inhibitors, with inhibition constants from the nanomolar to millimolar range. Acetazolamide inhibited the two enzymes with K(I)s in the range of 58-92 nM. The best anion inhibitors of Cd-R1 were thiocyanate, sulfamate and sulfamide, with K(I)s of 10-89 microM, whereas the best Zn-R1 anion inhibitors were sulfamate and sulfamide with K(I)s of 60-72 microM. These enzymes were only weakly inhibited by chloride, bromide or sulfate, main anion components of sea water, with inhibition constants in the range of 0.24-0.85 mM. Thus, similarly to CAs belonging to other classes, the zeta-class CA (with either cadmium or zinc ions at the active site) was inhibited by both anions and sulfonamides.

Carbonic anhydrase inhibitors. Regioselective synthesis of novel 1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamides and their inhibition of the human cytosolic isozymes I and II and transmembrane cancer-associated isozymes IX and XII.

A series of 1-substituted 1,4-dihydro-4-oxo-3-pyridinesulfonamide (2-16) have been synthesized and investigated as inhibitors of four isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is the cytosolic ubiquitous CA I and II, and cancer-associated isozymes CA IX and XII. Against the human isozymes hCA I the new compounds showed inhibition constants in the range of 1.09-12.1 microM, against hCA II in the range of 50.5-172 nM, against hCA IX in the range of 5.2-118 nM, and against hCA XII in the range of 8.7-381 nM, respectively. Compounds 2, 3, 5-9, 11, 13 and 14 showed excellent hCA IX inhibitory efficacy, with K(I)s = 5.2-11.0 nM, being much more effective as compared to the clinically used sulfonamides AAZ, MZA, EZA, DCP and IND (K(I)s = 24-50 nM). Compounds 2, 3, 5-9, 11 and 13 were also very effective hCA XII inhibitors (K(I)s = 8.7-45.2 nM) which are comparable or more effective than those clinically used EZA and DCP (K(I)s = 22 and 50 nM), respectively.

Nanoscale enzyme inhibitors: fullerenes inhibit carbonic anhydrase by occluding the active site entrance.

We investigated a series of derivatized fullerenes possessing alcohol, amine, and amino acid pendant groups as inhibitors of the zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1). We discovered that fullerenes bind CAs with submicromolar-low micromolar affinity, despite the fact that these compounds do not possess moieties normally associated with CA inhibitors such as the sulfonamides and their isosteres, or the coumarins. The 13 different mammalian CA isoforms showed a diverse inhibition profile with these compounds. By means of computational methods we assessed the inhibition mechanism as being due to occlusion of the active site entrance by means of the fullerene cage (possessing dimension of the same order of magnitude as the opening of the enzyme cavity, of 1nm). The pendant moieties to the fullerene cage make interactions with amino acid residues from the active site, among which His64, His94, His96, Val121, and Thr200. Fullerenes thus represent a totally new class of nanoscale CA inhibitors which may show applications for targeting physiologically relevant isoforms, such as the dominant CA II and the tumor-associated CA IX.

Carbonic anhydrase inhibitors: synthesis and inhibition of the human cytosolic isozymes I and II and transmembrane isozymes IX, XII (cancer-associated) and XIV with 4-substituted 3-pyridinesulfonamides.

A series of novel 4-substituted-3-pyridinesulfonamides (2-27 and 31-33) have been synthesized and investigated as inhibitors of five isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), that is, the cytosolic, ubiquitous isozymes CA I and II, and transmembrane isozymes CA IX, XII (cancer-associated) and XIV. Against the human isozymes hCA I, the new compounds showed inhibition constants in the range of 0.078-11.7 microM, against hCA II in the range of 9.9-140 nM, against hCA IX in the range of 4.6-313 nM, against hCA XII in the range of 3.4-21.6 nM, and against hCA XIV in the range of 50.9-160 nM, respectively. Compounds 4, 6, 7, 9, 11-14, 19, 20, 22-24, 26, 27, 31 and 32 showed excellent hCA IX inhibitory efficacy, with inhibition constants of 4.6-12.0 nM, being much more effective as compared to the clinically used sulfonamides AAZ, MZA, EZA, DCP and IND. 4-[N'-(6-chloro-7-cyano-1,1-dioxo-1,4,2-benzodithiazin-3-yl)hydrazino]-3-pyridinesulfonamide (31) is the prominent of the compounds due to its remarkable inhibitory activity toward hCA I (KIs=0.078 microM), hCA IX (KIs=7.2 nM) and hCA XII (KIs=3.4 nM).

Sulfonamide linked neoglycoconjugates--a new class of inhibitors for cancer-associated carbonic anhydrases.

The contribution of membrane-bound carbonic anhydrases (CAs) to hypoxic tumor growth and progression in cancer implicates cancer-associated CAs as a promising drug target for oncology. In this paper, we present a new class of sulfonamide-linked neoglycoconjugate that was designed to selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. We describe the application of novel, yet straightforward, chemistry toward the synthesis of inhibitors that comprise both S-glycosyl sulfenamides and S-glycosyl sulfonamides. We also present the CA inhibition profile of our new neoglycoconjugates, more specifically a library of 30 compounds (3-32) that were designed to optimize both SAR (structure-activity relationship) and SPR (structure-property relationship) characteristics. We show that our approach produces neutral, water-soluble, and potent inhibitors (K(i)s in the low nanomolar range) that target cancer-associated CAs.

Carbonic anhydrase inhibitors. Inhibition of transmembrane isoforms IX, XII, and XIV with less investigated anions including trithiocarbonate and dithiocarbamate.

An inhibition study of the transmembrane carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX, XII (tumor-associated), and XIV with anions such as stannate(IV), selenate(VI), tellurate(VI), perosmate(VIII), persulfate, pyrophosphate(V), pyrovanadate(V), tetraborate, persulfate, perrhenate(VII), perrutenate(VII), selenocyanate, iminodisulfonate, fluorosulfate, and trithiocarbonate is reported. Selenate, perosmate, and pyrophosphate were ineffective inhibitors, whereas most of these anions inhibited the three enzymes in the millimolar-submillimolar range. Trithiocarbonate and diethyldithiocarbamate were the best CA IX inhibitors (K(I)s of 1.4-9.7microM), but trithiocarbonate showed less affinity for CA XII and XIV (K(I)s of 0.12-0.66mM). N,N-Diethyldithiocarbamate was a low micromolar inhibitor also against CA XII and XIV (K(I)s of 1.0-1.1microM), suggesting that this new zinc-binding group (CS2-) may lead to efficient inhibitors targeting transmembrane CAs.