2-Substituted-4,7-dihydro-4-ethylpyrazolo[1,5-a]pyrimidin-7-ones alleviate LPS-induced inflammation by modulating cell metabolism via CD73 upon macrophage polarization.

Macrophage polarization towards the M1 phenotype under bacterial product-related exposure (LPS) requires a rapid change in gene expression patterns and cytokine production along with a metabolic rewiring. Metabolic pathways and redox reactions are such tightly connected, giving rise to an area of research referred to as immunometabolism. A role in this context has been paid to the master redox-sensitive regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and to the 5'-ectonucleotidase CD73, a marker related to macrophage metabolism rearrangement under pro-inflammatory conditions. In this light, a cell model of LPS-stimulated macrophages has been established and nine 4,7-dihydro-4-ethylpyrazolo[l,5-a]pyrimidin-7-ones with a potential anti-inflammatory effect have been administered. Our data highlight that two selected compounds (namely, 5 and 8) inhibit the LPS-induced Nrf2 nuclear translocation and ameliorate the activity rate of the antioxidant enzyme catalase. Additionally, the pyridine-containing compound (8) promotes the shift from the pro-inflammatory immunophenotype M1 to the pro-resolving M2 one, by downregulating CD80 and iNOS and by enhancing CD163 and TGFß1 expression. Most importantly, CD73 is modulated by these compounds as well as the lactate production. Our data demonstrate that pyrazolo[l,5-a]pyrimidine derivatives are effective as anti-inflammatory compounds. Furthermore, these pyrazolo[l,5-a]pyrimidines exert their action via CD73-related signaling and modulation of cell metabolism of activated macrophages.

First-in-Class Dual Targeting Compounds for the Management of Seizures in Glucose Transporter Type 1 Deficiency Syndrome.

The genetic disorder glucose transporter type 1 deficiency syndrome (GLUT1-DS) heavily affects the main intake of energy in tissues and determines the most relevant outcomes at the central nervous system (CNS) district, which is highly dependent on glucose. Herein, we report the design and development of a set of compounds bearing the glucosyl and galactosyl moieties. We assessed their ability to enhance the GLUT1 mediated glucose intake in non-small-cell lung cancer (NSCLC) cells and to inhibit the carbonic anhydrase (CA; EC 4.2.1.1) isoforms implicated in the physiopathology of uncontrolled seizures associated to epilepsy (i.e., I, II, IV, VA, VB, and XII). The binding mode of 8 in adduct with hCA II was determined by X-ray crystallography. Among the selected derivatives, compound 4b proved effective in suppressing the occurrence of uncontrolled seizures on the in vivo induced maximal electroshock (MES) model and thus gives sustainment of an unprecedently reported pharmacological approach for the management of GLUT1-DS associated diseases.

Seleno Containing Compounds as Potent and Selective Antifungal Agents.

Fungal promoted infections are becoming a severe health global emergency due to drug-resistant phenomena and zoonosis. This work investigated compounds bearing acyl-/selenoureido moieties and primary/secondary sulfonamide groups as novel antifungal agents acting through organism-directed selenium toxicity and inhibition of the newly emergent therapeutic target, the Carbonic Anhydrases (CAs; EC 4.2.1.1). Reported data clearly indicate that seleno-containing scaffolds with respect to the standard-of-care drugs showed appreciable antifungal activity, which was suppressed when the chalcogen was replaced with its cognate isosteric elements sulfur and oxygen. In addition, such compounds showed excellent selectivity against Malassezia pachydermatis over its related genus strains Malassezia furfur and Malassezia globosa. Safe cytotoxicity profiles on bovine kidney cells (MDBK) and human HaCat cells, as well as the shallow hemolytic activity on defibrinated sheep blood, allowed us to consider these compounds as up-and-coming novel antifungals.

Thiosemicarbazide-Substituted Coumarins as Selective Inhibitors of the Tumor Associated Human Carbonic Anhydrases IX and XII.

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.

Small Molecule Alkoxy Oriented Selectiveness on Human Carbonic Anhydrase II and IX Inhibition.

We report aryl sulfonamide inhibitors of human carbonic anhydrase (hCA; EC 4.2.1.1) enzymes containing short ureido alkoxy tails. The inhibition potency of such compounds was investigated in vitro on the major hCA isoforms (i.e. I, II, IX, and XII). A selection of the most potent inhibitory derivatives against the hCA IX isoform (i.e. 5a, 5c, and 6c) was studied, and their binding modes on either hCA II and IX-mimic isoform were assessed by X-ray crystallography on the corresponding ligand/protein adducts. This study adds to the field of developing hCA inhibitors at molecular level the critical interactions governing ligand selectivity.

Generation of Complex Syngeneic Liver Organoids from Induced Pluripotent Stem Cells to Model Human Liver Pathophysiology.

The study of human liver pathophysiology has been hampered for decades by the lack of easily accessible, robust, and representative in vitro models. The discovery of induced pluripotent stem cells (iPSCs)-which can be generated from patients' somatic cells, engineered to harbor specific mutations, and differentiated into hepatocyte-like cells-opened the way to more meaningful modeling of liver development and disease. Nevertheless, representative modeling of many complex liver conditions requires the recreation of the interplay between hepatocytes and nonparenchymal liver cells. Here we describe protocols we developed to generate and characterize complex human liver organoids composed of iPSC-derived hepatic, endothelial, and mesenchymal cells. With all cell types derived from the same iPSC population, such organoids reproduce the liver niche, allowing for the study of liver development and the modeling of complex inflammatory and fibrotic conditions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of human iPSCs into hepatic progenitor cells (hepatoblasts) Basic Protocol 2: Differentiation of human iPSCs into endothelial progenitor cells Support Protocol 1: Characterization of iPSC-derived endothelial progenitor cells Basic Protocol 3: Differentiation of human iPSCs into mesenchymal progenitor cells Support Protocol 2: Characterization of iPSC-derived mesenchymal progenitor cells Basic Protocol 4: Generation of complex syngeneic liver organoids.

Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes.

Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.

Heterologous expression and biochemical characterisation of the recombinant ß-carbonic anhydrase (MpaCA) from the warm-blooded vertebrate pathogen malassezia pachydermatis.

Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO2/HCO3-/pH-sensing in fungi is triggered by HCO3- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the ß-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.

Inhibitory Effects of Sulfonamide Derivatives on the ß-Carbonic Anhydrase (MpaCA) from Malassezia pachydermatis, a Commensal, Pathogenic Fungus Present in Domestic Animals.

Fungi are exposed to various environmental variables during their life cycle, including changes in CO2 concentration. CO2 has the potential to act as an activator of several cell signaling pathways. In fungi, the sensing of CO2 triggers cell differentiation and the biosynthesis of proteins involved in the metabolism and pathogenicity of these microorganisms. The molecular machineries involved in CO2 sensing constitute a promising target for the development of antifungals. Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial enzymes in the CO2 sensing systems of fungi, because they catalyze the reversible hydration of CO2 to proton and HCO3-. Bicarbonate in turn boots a cascade of reactions triggering fungal pathogenicity and metabolism. Accordingly, CAs affect microorganism proliferation and may represent a potential therapeutic target against fungal infection. Here, the inhibition of the unique ß-CA (MpaCA) encoded in the genome of Malassezia pachydermatis, a fungus with substantial relevance in veterinary and medical sciences, was investigated using a series of conventional CA inhibitors (CAIs), namely aromatic and heterocyclic sulfonamides. This study aimed to describe novel candidates that can kill this harmful fungus by inhibiting their CA, and thus lead to effective anti-dandruff and anti-seborrheic dermatitis agents. In this context, current antifungal compounds, such as the azoles and their derivatives, have been demonstrated to induce the selection of resistant fungal strains and lose therapeutic efficacy, which might be restored by the concomitant use of alternative compounds, such as the fungal CA inhibitors.

Privileged scaffolds in medicinal chemistry: Studies on pyrazolo[1,5-a]pyrimidines on sulfonamide containing Carbonic Anhydrase inhibitors.

We report for the first time a small series of compounds endowed in vitro with inhibitory properties for the human (h) expressed Carbonic Anhydrase (CAs, E.C. 4.2.1.1) enzymes of physiological interest (i.e. I, II, VA, IX and XII) and bearing the pyrazolo[1,5-a]pyrimidine (PP) scaffold at the tail section. Among the series reported, 1a-3a, 7a, 8a, 1b and 2b resulted effective ligands and with good selectivities for the hCAs II, IX or XII. In consideration of the nearly matching KI values of 7a for both the hCA II and IX (i.e. 26.4 and 23.0 nM respectively) we explored its binding mode within the CA IX mimic isoform by means of X-ray crystal experiments on the corresponding adduct.

Structural Insights into Schistosoma mansoni Carbonic Anhydrase (SmCA) Inhibition by Selenoureido-Substituted Benzenesulfonamides.

Tegumental carbonic anhydrase from the worm Schistosoma mansoni (SmCA) is considered a new anti-parasitic target because suppressing its expression interferes with schistosome metabolism and virulence. Here, we present the inhibition profiles of selenoureido compounds on recombinant SmCA and resolution of the first X-ray crystal structures of SmCA in adduct with a selection of such inhibitors. The key molecular features of such compounds in adduct with SmCA were obtained and compared to the human isoform hCA II, in order to understand the main structural factors responsible for enzymatic affinity and selectivity. Compounds that more specifically inhibited the schistosome versus human enzymes were identified. The results expand current knowledge in the field and pave the way for the development of more potent antiparasitic agents in the near future.

Tellurides bearing benzensulfonamide as carbonic anhydrase inhibitors with potent antitumor activity.

We evaluated in vitro a series of telluride containing compounds bearing the benzenesulfonamide group, as effective inhibitors of the physiologically relevant human (h) expressed Carbonic Anhydrase (CA; EC 4.2.1.1) enzymes I, II, IV VII and IX. The potent effects of such compounds against the tumor-associated hCA IX being low nanomolar inhibitors (KI 2.2 to 2.9 nM) and with good selectivity over the ubiquitous hCA II, gave the possibility to evaluate their lethal effect in vitro against a breast cancer cell line (MDA-MB-231). Among the series, both compounds 3a and 3g induced significant toxic effects against tumor cells after 48 h incubation. Under normoxic condition 3a showed high efficacy killing over 94% of tumor cells at 1 µM, and derivative 3g reached the tumor cell viability under the 5% at 10 µM. In hypoxic condition, these two compounds showed less effective although retained excellent cancer cell killer. These unusual features make them interesting lead compounds acting as antitumor agents also in tumor types not dependent from hCA IX overexpression.

Taurultams incorporating arylsulfonamide: First in vitro inhibition studies of α-, ß- and γ-class Carbonic Anhydrases from Vibrio cholerae and Burkholderia pseudomallei.

A new series of taurultambenzenesulfonamides 1-17 were prepared and considered for their inhibitory activity in vitro against the Carbonic Anhydrases from Vibrio cholerae (VchCA-α, VchCA-ß and VchCA-γ) and Burkholderia pseudomallei (BpsCA-ß and BpsCA-γ). Among the compounds tested, derivatives 4, 5, 7, 10, 12, and 16 resulted in highly effective VchCAα inhibitors (KI values spanning within the 6.1-9.6 nM range) and endowed with excellent Selectivity Indexes (SIs; KI VchCA-α/KI hCA II) all comprised between 0.04 and 0.09. Potent in vitro inhibitors for the BpsCA-γ were also identified (KIs of 18.9-19.5 nM). The results here reported may represent the blueprint for the future development of a new generation of CA-based antibiotics integrated with free of resistance mechanisms of action adopted from known drugs.

Handling drug-target selectivity: A study on ureido containing Carbonic Anhydrase inhibitors.

Here we report the synthesis of a series of taurine substituted sulfonamide derivatives 1-29 having the ureido moiety installed at the tail section as selective inhibitors of the tumor associated human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) IX and XII. The series was deeply investigated for their kinetic features which demonstrated a strong dependence on the ureido moiety. High resolution X-ray crystallographic investigation on selected ligand adducts complexed with hCA II and hCA IX-mimic revealed a strong correlation between the ureido moiety and the amino acid residues Q92 and Q67 in both the hCA II and hCA IX-mimic, contributing to highly stabilized ligand-protein complex.

Azidothymidine "Clicked" into 1,2,3-Triazoles: First Report on Carbonic Anhydrase-Telomerase Dual-Hybrid Inhibitors.

Cancer cells rely on the enzyme telomerase (EC 2.7.7.49) to promote cellular immortality. Telomerase inhibitors (i.e., azidothymidine) can represent promising antitumor agents, although showing high toxicity when administered alone. Better outcomes were observed within a multipharmacological approach instead. In this context, we exploited the validated antitumor targets carbonic anhydrases (CAs; EC 4.2.1.1) IX and XII to attain the first proof of concept on CA-telomerase dual-hybrid inhibitors. Compounds 1b, 7b, 8b, and 11b showed good in vitro inhibition potency against the CAs IX and XII, with KI values in the low nanomolar range, and strong antitelomerase activity in PC-3 and HT-29 cells (IC50 values ranging from 5.2 to 9.1 µM). High-resolution X-ray crystallography on selected derivatives in the adduct with hCA II as a model study allowed to determine their binding modes and thus to set the structural determinants necessary for further development of compounds selectively targeting the tumoral cells.

X-ray crystallography of Epacadostat in adduct with Carbonic Anhydrase IX.

Epacadostat (EPA), a new and promising anti-cancer small molecule is firmly established as selective inhibitor of the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). The X-Ray structure of the human CA IX mimic in complex with EPA is investigated here for the first time and compared to previously reported EPA-CA II adduct. The structural information obtained are all in agreement with the in vitro kinetic data which accounted for a selective inhibition of the CA IX over the CA II isoform.

Bioisosteric Development of Multitarget Nonsteroidal Anti-Inflammatory Drug-Carbonic Anhydrases Inhibitor Hybrids for the Management of Rheumatoid Arthritis.

Multitarget nonsteroidal anti-inflammatory drug (NSAID)-carbonic anhydrase inhibitor (CAI) agents for the management of rheumatoid arthritis are reported. The evidence of the plasma stability of the amide-linked hybrids previously reported prompted us to investigate their pain-relieving mechanism of action. A bioisosteric amide to ester substitution yielded a series of derivatives showing potent target CAs inhibition and to undergo cleavage in rat or human plasma depending on the NSAID portion. A selection of derivatives were assayed in vitro to indirectly evaluate their effect on COX-1 and COX-2. MD simulations demonstrated that the entire hybrids are also able to efficiently bind the COX active site. In a rat model of RA, the most promising derivative (5c) showed major antihyperalgesic action compared with the equimolar coadministration of the single agents. The gathered data provided new insights on the action mechanism of these multitarget compounds, which induce markedly improved pain relief compared with the parent NSAIDs.

N-aryl-N'-ureido-O-sulfamates: Potent and selective inhibitors of the human Carbonic Anhydrase VII isoform with neuropathic pain relieving properties.

Herein we report for the first time an efficient synthetic procedure for the preparation of N-aryl-N'-ureido-O-sulfamates (AUSs) as a new class of Carbonic Anhydrase Inhibitors (CAIs). The compounds were tested for the inhibition of several human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) isoforms. Interesting inhibition activity and high selectivity against CA VII and XII versus CA I and II, with KIs in the low nanomolar range, were observed. Molecular modeling studies allowed us to decipher the structural features underpinning the selective inhibitory profile of AUSs towards isoforms CAs VII and XII. A selection of sulfamates showed promising neuropathic pain modulating effects in an in vivo animal model of oxaliplatin induced pain.

Polypharmacology of epacadostat: a potent and selective inhibitor of the tumor associated carbonic anhydrases IX and XII.

Epacadostat (EPA), a selective indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor, has been investigated in vitro as a human (h) Carbonic Anhydrase Inhibitor (CAI). The kinetic data clearly show, for the first time, EPA to be a highly effective and selective inhibitor for the tumor-associated isoforms hCA IX/XII. We report the high resolution X-ray crystal structure of the EPA-hCA II adduct, and assessed its binding mode to CA IX/XII by means of computational techniques. EPA may exert antitumor effects also due to the potent inhibition of the tumor-associated CAs.