Discovery and characterization of potent spiro-isoxazole-based cereblon ligands with a novel binding mode.

The vast majority of current cereblon (CRBN) ligands is based on the thalidomide scaffold, relying on glutarimide as the core binding moiety. With this architecture, most of these ligands inherit the overall binding mode, interactions with neo-substrates, and thereby potentially also the cytotoxic and teratogenic properties of the parent thalidomide. In this work, by incorporating a spiro-linker to the glutarimide moiety, we have generated a new chemotype that exhibits an unprecedented binding mode for glutarimide-based CRBN ligands. In total, 16 spirocyclic glutarimide derivatives incorporating an isoxazole moiety were synthesized and tested for different criteria. In particular, all ligands showed a favorable lipophilicity, and several were able to outperform the binding affinity of thalidomide as a reference. In addition, all compounds showed favorable cytotoxicity profiles in myeloma cell lines and human peripheral blood mononuclear cells. The novel binding mode, which we determined in co-crystal structures, provides explanations for these improved properties: The incorporation of the spiro-isoxazole changes both the conformation of the glutarimide moiety within the canonical tri-trp pocket and the orientation of the protruding moiety. In this new orientation it forms additional hydrophobic interactions and is not available for direct interactions with the canonical neo-substrates. We therefore propose this chemotype as an attractive building block for the design of PROTACs.

New Biocides Based on N4-Alkylcytidines: Effects on Microorganisms and Application for the Protection of Cultural Heritage Objects of Painting.

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N4-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N4-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N4-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N4-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection.

Carbonic Anhydrase Inhibitors Induce Ferroptosis through Inhibition of AKT/FTH1 Signaling in Ewing Sarcoma Tumor Cells.

Ewing sarcoma (ES) is one of the most frequent types of malignant tumors among children. The active metabolic state of ES cells presents a new potential target for therapeutic interventions. As a primary regulator of cellular homeostasis, carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as promising molecular targets for the development of anticancer drugs. Within the present study, we tested the commercial drug acetazolamide and our previously discovered inhibitors to target the CAII isoform, which was overexpressed and positively correlated with ES patient relapse. We employed molecular biology tests to identify effective inhibitors of CAII that can induce ferroptosis by downregulating FTH1 expression in ES cells. In vitro, we have also demonstrated their ability to reduce cell proliferation, decrease invasion, and induce apoptosis- or autophagy-related cell death. Using Western blotting, we confirmed the induction of cathepsin B in cells treated with CA inhibitors. It was found that the suppression of cathepsin B expression during the treatment reduces the anticancer efficacy of selected CAII inhibitors. These experiments highlighted profound antitumor activity of CAII inhibitors attributive to their remarkable ability to trigger ferroptosis in Ewing sarcoma cells without causing substantial host damage. The obtained results suggest that cytosolic CAII may be a prospective target for ES treatment, and CAII inhibitors can be considered as potential single-agent or combination antitumor agents to be used in the treatment of ES.

Substrate-Controlled Three-Component Synthesis of Diverse Fused Heterocycles.

A chemoselective strategy toward a variety of fused heterocyclic scaffolds relying on a three-component condensation of heterocyclic ketene aminals (HKAs) or corresponding thioaminals with aryl glyoxals and cyclic 1,3-dicarbonyl compounds has been developed and explored. Depending on the applied combination of substrates, the strategy can be tuned to provide straightforward access to imidazo[1,2-a]quinoline, pyrrolo[1,2-a]imidazole, and pyrrolo[2,1-b]thiazole frameworks.

Facile access to 3-sulfonylquinolines via Knoevenagel condensation/aza-Wittig reaction cascade involving ortho-azidobenzaldehydes and ß-ketosulfonamides and sulfones.

Quinoline-based sulfonyl derivatives, and especially sulfonamides, are relevant and promising structures for drug design. We have developed a new convenient protocol for the synthesis of 3-sulfonyl-substituted quinolines (sulfonamides and sulfones). The approach is based on a Knoevenagel condensation/aza-Wittig reaction cascade involving o-azidobenzaldehydes and ketosulfonamides or ketosulfones as key building blocks. The protocol is appropriate for both ketosulfonyl reagents and α-sulfonyl-substituted alkyl acetates providing the target quinoline derivatives in good to excellent yields.

Versatile Diazomethane Sulfonamide for Expedited Exploration of Azole-Based Carbonic Anhydrase Inhibitors via [3+2] Cycloaddition.

A newly introduced diazo reagent, 1-diazo-N,N-bis(4-methoxybenzyl)methanesulfonamide, enables access to a range of azole-based primary sulfonamides via [3+2] cycloaddition followed by protecting group removal. Such compounds are representative of the sulfonamide chemical space highly relevant but hitherto not investigated in the context of inhibition of therapeutically relevant isoforms of carbonic anhydrase enzyme. Using this reagent, three sets of primary sulfonamides based on pyrazole, 1,2,3-triazole and tetrazole cores were synthesized and profiled for inhibition of tumor-associated hCA IX and XII isoforms as well as abundant cytosolic hCA I and II isoforms. Using virtual library design and docking prioritization tool of the Schrödinger suite, one of the promising leads was evolved into a dual hCA IX/XII inhibitor with excellent selectivity over off-target hCA I and II. The new synthetic strategy to access azole-based primary sulfonamides will support the discovery of novel, isoform-selective inhibitors of carbonic anhydrase within the poorly explored azole chemical space.

A Series of Trifluoromethylisoxazolyl- and Trifluoromethylpyrazolyl- Substituted (Hetero)aromatic Sulfonamide Carbonic Anhydrase Inhibitors: Synthesis, and Convenient Prioritization Workflow for Further In Vivo Studies.

AIMS: To synthesize novel sulfonamide inhibitors of carbonic anhydrase and develop in vitro prioritization workflow to select compounds for in vivo evaluation. BACKGROUND: Carbonic anhydrase (CA) inhibitors gain significant attention in the context of drug discovery research for glaucoma, hypoxic malignancies, and bacterial infections. In previous works, we have successfully used direct sulfochlorination approach to develop diverse heterocyclic primary sulfonamides with remarkable activity and selectivity against therapeutically relevant CA isoforms. OBJECTIVE: Synthesis and investigation of the CA inhibitory properties of novel trifluoromethylisoxazolyl- and trifluoromethylpyrazolyl-substituted (hetero)aromatic sulfonamides. METHODS: Thirteen trifluoromethylisoxazolyl- and thirteen trifluoromethylpyrazolyl-substituted (hetero) aromatic sulfonamides were synthesized by direct sulfochlorination of hydroxyisoxazolines and pyrazoles followed by reaction with ammonia. The compound structures were confirmed by 1H and 13C NMR as well as element analysis. The obtained compounds were evaluated, using the CA esterase activity assay, for their potential to block the catalytic activity of bovine CA (bCA). RESULTS: Eight most potent compounds selected based on the esterase activity assay data were tested for direct affinity to the enzyme using the thermal shift assay (TSA). These compounds displayed Kd values (measured by TSA) in the double-digit nanomolar range, thus showing comparable activity to the reference drug acetazolamide. CONCLUSION: Coupling the bCA esterase activity assay with thermal shift assay represents a streamlined and economical strategy for the prioritization of sulfonamide CA inhibitors for subsequent evaluation in vivo.

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. II. Quantitative analysis of multi-state kinetic networks.

Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are ideally suited to resolve the structural dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with pseudo-static molecules in similar conformational states with undistinguishable Förster Resonance Energy Transfer (FRET) efficiencies. To address these problems, we developed a quantitative integrative analysis framework that combines the information from FRET-lines that relate average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms, fluorescence decays obtained by time-correlated single-photon-counting, photon distribution analysis of the intensities, and fluorescence correlation spectroscopy. Individually, these methodologies provide ambiguous results for the characterization of dynamics in complex kinetic networks. However, the global analysis approach enables accurate determination of the number of states, their kinetic connectivity, the transition rate constants, and species fractions. To challenge the potential of smFRET experiments for studying multi-state kinetic networks, we apply our integrative framework using a set of synthetic data for three-state systems with different kinetic connectivity and exchange rates. Our methodology paves the way toward an integrated analysis of multiparameter smFRET experiments that spans all dimensions of the experimental data. Finally, we propose a workflow for the analysis and show examples that demonstrate the usefulness of this toolkit for dynamic structural biology.

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τD and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E-τD diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines.

5-(Sulfamoyl)thien-2-yl 1,3-oxazole inhibitors of carbonic anhydrase II with hydrophilic periphery.

Hydrophilic derivatives of an earlier described series of carbonic anhydrase inhibitors have been designed, prepared and profiled against a panel of carbonic anhydrase isoforms, including the glaucoma-related hCA II. For all hydrophilic derivatives, computational prediction of intraocular permeability routes showed the predominance of conjunctival rather than corneal absorption. The potentially reactive primary or secondary amine periphery of these compounds makes them suitable candidates for bioconjugation to polymeric drug carriers. As was shown previously, the most active hCA II inhibitor is efficacious in alleviating intraocular pressure in normotensive rabbits with efficacy matching that of dorzolamide.

Diversely substituted sulfamides for fragment-based drug discovery of carbonic anhydrase inhibitors: synthesis and inhibitory profile.

A series of sulfamide fragments has been synthesised and investigated for human carbonic anhydrase inhibition. One of the fragments showing greater selectivity for cancer-related isoforms hCA IX and XII was co-crystalized with hCA II showing significant potential for fragment periphery evolution via fragment growth and linking. These opportunities will be identified in the future via the screening of this fragment structure for co-operative carbonic anhydrase binding with other structurally diverse fragments.[Figure: see text].

Synthesis, Structure, and Antiproliferative Action of 2-Pyridyl Urea-Based Cu(II) Complexes.

Relying on a recently suggested protocol that furnishes convenient access to variously substituted 2-pyridyl ureas, twelve hitherto unknown Cu(II) complexes have been synthesized in the present work and their structures were evaluated by elemental analysis, HRMS, IR spectroscopy, and X-ray diffraction study. Two structural motifs ([Cu(L)2Cl]+[Cl]- or (Cu(L)2Cl2) depending on the substitution pattern on the 2-pyridine fragment were revealed. In addition, antiproliferative action of the obtained compounds have been investigated against lung cancer cell lines (A549, NCI-H460, NCI-H1975), and healthy WI-26 VA4 cells were used to monitor non-specific cytotoxicity. Two nitro-group substituted complexes Cu(U3)2Cl2 (IC50 = 39.6 ± 4.5 µM) and Cu(U11)2Cl2 (IC50 = 33.4 ± 3.8 µM) demonstrate enhanced activity against the drug resistant NCI-H1975 cells with moderate selectivity toward normal WI-26 VA4 cells. The antiproliferative mechanism of cell death underlying the growth inhibitory effect of the synthesized complexes was studied via additional experiments, including the cell cycle analysis and the apoptosis induction test. Reassuringly, certain 2-pyridyl urea-based Cu(II) complexes exerted cell line-specific antiproliferative effect which renders them valuable starting points for further unveiling the anticancer potential of this class of coordination compounds.

Carbonic Anhydrase IX Inhibitors as Candidates for Combination Therapy of Solid Tumors.

Combination therapy is becoming imperative for the treatment of many cancers, as it provides a higher chance of avoiding drug resistance and tumor recurrence. Among the resistance-conferring factors, the tumor microenvironment plays a major role, and therefore, represents a viable target for adjuvant therapeutic agents. Thus, hypoxia and extracellular acidosis are known to select for the most aggressive and resilient phenotypes and build poorly responsive regions of the tumor mass. Carbonic anhydrase (CA, EC 4.2.1.1) IX isoform is a surficial zinc metalloenzyme that is proven to play a central role in regulating intra and extracellular pH, as well as modulating invasion and metastasis processes. With its strong association and distribution in various tumor tissues and well-known druggability, this protein holds great promise as a target to pharmacologically interfere with the tumor microenvironment by using drug combination regimens. In the present review, we summarized recent publications revealing the potential of CA IX inhibitors to intensify cancer chemotherapy and overcome drug resistance in preclinical settings.

Mucoadhesive properties of nanogels based on stimuli-sensitive glycosaminoglycan-graft-pNIPAAm copolymers.

Mucoadhesive formulations capable of situ gelation are promising for improving ocular drug delivery. Here we investigated two types of nanogels based on anionic glycosaminoglycans with grafted thermo-responsive poly(N-isopropylacrylamide) chains. One type of nanogels were formed by thermo-induced gelling of heparin-graft-poly(N-isopropylacrylamide) and chondroitin sulfate-graft-poly(N-isopropylacrylamide) copolymers. Another type of nanogels was based on the same copolymers, but terminal groups of thermosensitive macromolecular chains were modified to form covalent disulfide cross-links. All types of nanogels were studied towards their ability to encapsulate and release model drug - dexamethasone. Mucoadhesivity of both thermo-gelled and covalently cross-linked polymeric systems, as well as their ability to interact with dexamethasone, was assessed by microscale thermophoresis (MST). Mucoadhesion properties were also evaluated by isothermal titration calorimetry (ITC), which were in good correlation with MST data. The presence of disulfide linkages and thiol groups were shown to favor improved binding of cross-linked nanogels to mucin. Moreover, in vivo intraocular pressure studies showed that presence of polymers in solution can alter the ocular absorption of carbonic anhydrase inhibitor from eyedrops. The pharmacological effect was in line with mucoadhesive properties of these copolymers.

Investigation of 3-sulfamoyl coumarins against cancer-related IX and XII isoforms of human carbonic anhydrase as well as cancer cells leads to the discovery of 2-oxo-2H-benzo[h]chromene-3-sulfonamide - A new caspase-activating proapoptotic agent.

Herein we report the synthesis of a set of seventeen 3-sulfonamide substituted coumarin derivatives. Prepared compounds were tested in vitro for inhibition of four physiologically relevant isoforms of the metalloenzyme human carbonic anhydrase (hCA, EC 4.2.1.1). Several coumarin sulfonamides displayed low nanomolar KI values against therapeutically relevant hCA II, IX, and XII, whereas they did not potently inhibit hCA I. Some of these compounds exerted a concentration-dependent antiproliferative action toward RT4 human bladder cancer and especially A431 human epidermoid carcinoma cell lines. In the meantime, the viability of non-tumorigenic hTERT immortalized human foreskin fibroblast cell line Bj-5ta was not significantly affected by the obtained derivatives. Interestingly, compound 10q (2-oxo-2H-benzo [h]chromene-3-sulfonamide) showed a profound and selective dose-dependent inhibition of A431 cell growth with low nanomolar IC50 values. We demonstrated that 10q possessed a concentration-dependent apoptosis induction activity associated with caspase 3/7 activation in cancer cells. As carbonic anhydrase isoforms in question were not potently inhibited by this compound, its antiproliferative effects likely involve other mechanisms, such as DNA intercalation. Compound 10q clearly represents a viable lead for further development of new-generation anticancer agents.

Biochemical profiling of anti-HIV prodrug Elsulfavirine (Elpida®) and its active form VM1500A against a panel of twelve human carbonic anhydrase isoforms.

The non-nucleoside reverse transcriptase inhibitor VM1500A is approved for the treatment of HIV/AIDS in its N-acyl sulphonamide prodrug form elsulfavirine (Elpida®). Biochemical profiling against twelve human carbonic anhydrase (CA, EC 4.2.1.1) isoforms showed that while elsulfavirine was a weak inhibitor of all isoforms, VM1500A potently and selectively inhibited human (h) hCA VII isoform, a proven target for the therapy of neuropathic pain. The latter is a common neurologic complication of HIV infection and we hypothesise that by using Elpida® in patients may help alleviate this debilitating symptom.

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 in vitro 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.

Antimicrobial Activity of 5-membered Nitroheteroaromatic Compounds beyond Nitrofurans and Nitroimidazoles: Recent Progress.

The last decade has been characterized by the development and approval of pretomanid and delamanid, which are nitroimidazole based drugs for multidrug -resistant tuberculosis. This attracted renewed attention to the nitroheterocyclic scaffolds as a source of safe and efficient antimicrobial agents. While the primary focus is still on nitrofurans and nitroimidazoles, well known as bioreducible prodrugs, a number of studies have been published on other 5-membered nitroheteroaromatic compounds. The latter not only show promising antimicrobial activity but also demonstrate modes of action different from the conventional reductive activation of the nitro group. Considering the potential of these efforts to impact the continuing race against drug-resistant pathogens, herein we review non-furan/imidazole-based 5-membered nitroheteroaromatics investigated as antimicrobial agents in 2010-2020.

Radiotracers for positron emission tomography (PET) targeting tumour-associated carbonic anhydrase isoforms.

The tumour-associated, cell membrane-bound isoforms IX and XII of human carbonic anhydrase (CA, EC 4.2.1.1) are overexpressed in cancer cells contributing to the hypoxic tumour pH/metabolism regulating machinery and as thus, can serve as markers of malignant neoplastic tissue. Inhibitors of CAs can be employed both for the treatment of hypoxic tumours and in the design of radiotracers for positron emission tomography and imaging of such cancers. The present review provides a comprehensive summary of the progress achieved to-date in the field of developing PET-tracers based on monoclonal antibodies, biomolecules, and small-molecule ligands of CA IX and XII.