Exploring the Redox and pH Dimension of Carbonic Anhydrases in Cancer: A Focus on Carbonic Anhydrase 3.

Significance: Both redox and pH are important regulatory processes that underpin cell physiological functions, in addition to influencing cancer cell development and tumor progression. The thioredoxin (Trx) and glutathione redox systems and the carbonic anhydrase (CA) proteins are considered key regulators of cellular redox and pH, respectively, with components of the Trx system and CAs regarded as cancer therapeutic targets. However, the redox and pH axis in cancer cells is an underexplored topic of research. Recent Advances: Structural studies of a CA family member, CA3, localized two of its five cysteine residues to the protein surface. Redox-regulated modifications to CA3 have been identified, including glutathionylation. CA3 has been shown to bind to other proteins, including B cell lymphoma-2-associated athanogene 3, and squalene epoxidase, which can modulate autophagy and proinflammatory signaling, respectively, in cancer cells. Critical Issues: CA3 has also been associated with epithelial-mesenchymal transition processes, which promote cancer cell metastasis, whereas CA3 overexpression activates the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, which upregulates cell growth and inhibits autophagy. It is not yet known if CA3 modulates cancer progression through its reported antioxidant functions. Future Directions: CA3 is one of the least studied CA isozymes. Further studies are required to assess the cellular antioxidant role of CA3 and its impact on cancer progression. Identification of other binding partners is also required, including whether CA3 binds to Trx in human cells. The development of specific CA3 inhibitors will facilitate these functional studies and allow CA3 to be investigated as a cancer therapeutic target.

Fragment-based drug discovery campaigns guided by native mass spectrometry.

Native mass spectrometry (nMS) is well established as a biophysical technique for characterising biomolecules and their interactions with endogenous or investigational small molecule ligands. The high sensitivity mass measurements make nMS particularly well suited for applications in fragment-based drug discovery (FBDD) screening campaigns where the detection of weakly binding ligands to a target biomolecule is crucial. We first reviewed the contributions of nMS to guiding FBDD hit identification in 2013, providing a comprehensive perspective on the early adoption of nMS for fragment screening. Here we update this initial progress with a focus on contributions of nMS that have guided FBDD for the period 2014 until end of 2023. We highlight the development of nMS adoption in FBDD in the context of other biophysical fragment screening techniques. We also discuss the roadmap for increased adoption of nMS for fragment screening beyond soluble proteins, including for guiding the discovery of fragments supporting advances in PROTAC discovery, RNA-binding small molecules and covalent therapeutic drug discovery.

Thymidine Kinase-Independent Click Chemistry DNADetect Probes for DNA Proliferation Assessment in Malaria Parasites.

Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Native Mass Spectrometry: Insights and Opportunities for Targeted Protein Degradation.

Native mass spectrometry (nMS) is one of the most powerful biophysical methods for the direct observation of noncovalent protein interactions with both small molecules and other proteins. With the advent of targeted protein degradation (TPD), nMS is now emerging as a compelling approach to characterize the multiple fundamental interactions that underpin the TPD mechanism. Specifically, nMS enables the simultaneous observation of the multiple binary and ternary complexes [i.e., all combinations of E3 ligase, target protein of interest, and small molecule proximity-inducing reagents (such as PROteolysis TArgeting Chimeras (PROTACs) and molecular glues)], formed as part of the TPD equilibrium; this is not possible with any other biophysical method. In this paper we overview the proof-of-concept applications of nMS within the field of TPD and demonstrate how it is providing researchers with critical insight into the systems under study. We also provide an outlook on the scope and future opportunities offered by nMS as a core and agnostic biophysical tool for advancing research developments in TPD.

The Glitazone Class of Drugs as Carbonic Anhydrase Inhibitors-A Spin-Off Discovery from Fragment Screening.

The approved drugs that target carbonic anhydrases (CA, EC 4.2.1.1), a family of zinc metalloenzymes, comprise almost exclusively of primary sulfonamides (R-SO2NH2) as the zinc binding chemotype. New clinical applications for CA inhibitors, particularly for hard-to-treat cancers, has driven a growing interest in the development of novel CA inhibitors. We recently discovered that the thiazolidinedione heterocycle, where the ring nitrogen carries no substituent, is a new zinc binding group and an alternate CA inhibitor chemotype. This heterocycle is curiously also a substructure of the glitazone class of drugs used in the treatment options for type 2 diabetes. Herein, we investigate and characterise three glitazone drugs (troglitazone 11, rosiglitazone 12 and pioglitazone 13) for binding to CA using native mass spectrometry, protein X-ray crystallography and hydrogen-deuterium exchange (HDX) mass spectrometry, followed by CA enzyme inhibition studies. The glitazone drugs all displayed appreciable binding to and inhibition of CA isozymes. Given that thiazolidinediones are not credited as a zinc binding group nor known as CA inhibitors, our findings indicate that CA may be an off-target of these compounds when used clinically. Furthermore, thiazolidinediones may represent a new opportunity for the development of novel CA inhibitors as future drugs.

Carbonic anhydrase XII inhibition overcomes P-glycoprotein-mediated drug resistance: a potential new combination therapy in cancer.

Intrinsic or acquired resistance to chemotherapy is a major hurdle in the treatment of cancer. One of the key mechanisms of resistance is the overexpression of the drug efflux transporter P-glycoprotein (Pgp). Pgp overexpression renders a large number of mechanistically unrelated chemotherapies ineffective. Targeting Pgp inhibition directly to overcome drug resistance, although conceptually and mechanistically attractive, has not translated to the clinic, in part because Pgp also has a critical protective function in many healthy tissues. It was recently discovered that carbonic anhydrase XII (CA XII), an enzyme associated with pH regulation in cancer, is co-expressed and co-located with Pgp in drug resistant cancer cells. CA XII is also upregulated by hypoxia, which is another microenvironmental factor that contributes to drug resistance. Here, we review findings that demonstrate modulation of CA XII may offer a promising new approach towards overcoming the longstanding hurdle of drug resistance and therapy failure against solid cancers. This review covers the use of CA XII inhibitors, both small molecule and antibody, in combination with chemotherapeutics that are substrates for Pgp. This combination therapy approach restores the efficacy of chemotherapy in resistant cells and offers a potential new therapeutic window to re-examine the targeting of Pgp as a safe, effective, and novel anticancer strategy.

The Key Glycolytic Enzyme Phosphofructokinase Is Involved in Resistance to Antiplasmodial Glycosides.

Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. Here, we show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). Flux through the upper glycolysis pathway was significantly reduced in PS-3-resistant parasites, which was associated with reduced ATP levels but increased flux into the pentose phosphate pathway. PS-3 may directly or indirectly target enzymes in these pathways, as PS-3-treated parasites had elevated levels of glycolytic and tricarboxylic acid (TCA) cycle intermediates. PS-3 resistance also led to reduced MEP pathway intermediates, and PS-3-resistant parasites were hypersensitive to the MEP pathway inhibitor, fosmidomycin. Overall, this study suggests that PS-3 disrupts core pathways in central carbon metabolism, which is compensated for by mutations in PfPFK9, highlighting a novel metabolic drug resistance mechanism in P. falciparumIMPORTANCE Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway.

Investigation of pyrimidine nucleoside analogues as chemical probes to assess compound effects on the proliferation of Trypanosoma cruzi intracellular parasites.

Trypanosoma cruzi parasites utilise de novo pyrimidine biosynthesis to produce DNA and survive within mammalian host cells. This pathway can be hijacked to assess the replication of intracellular parasites with the exogenous addition of a DNA specific probe. To identify suitable probe compounds for this application, a collection of pyrimidine nucleoside analogues was assessed for incorporation into T. cruzi intracellular amastigote DNA using image-based technology and script-based analysis. Associated mammalian cell toxicity of these compounds was also determined against both the parasite host cells (3T3 cells) and HEK293 cells. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into parasite DNA was the most effective of the probes tested, with minimal growth inhibition observed following either two or four hours EdU exposure. EdU was subsequently utilised as a DNA probe, followed by visualisation with click chemistry to a fluorescent azide, to assess the impact of drugs and compounds with previously demonstrated activity against T. cruzi parasites, on parasite replication. The inhibitory profiles of these molecules highlight the benefit of this approach for identifying surviving parasites post-treatment in vitro and classifying compounds as either fast or slow-acting. F-ara-EdU resulted in <50% activity observed against T. cruzi amastigotes following 48 hours incubation, at 73 µM. Collectively, this supports the further development of pyrimidine nucleosides as chemical probes to investigate replication of the parasite T. cruzi.

Synthesis of 5-Alkynyl Substituted 2'-Arabinosyl 2'-Halogenated Uridine Nucleosides.

This unit describes the detailed preparation of 5-alkynyl-2'-halogenated arabinosyl uridine nucleosides (2'-halo-ara-EdU) from uridine. These compounds were synthesized as prospective chemical probes for the detection of DNA synthesis in proliferating cells. Currently, this is the only synthetic methodology reported to access these compounds. The key to success of the synthetic approach was to employ a 3-N-nitro-protecting group to stabilize the required 2'-triflate nucleoside precursor toward nucleophilic substitution. Several synthetic challenges were overcome to accommodate the combination of a 5-alkyne and 3-N-nitro functional group, including facile introduction and removal of the N-nitro group, and removal of the sugar acetyl groups under acidic conditions. © 2019 by John Wiley & Sons, Inc.

Oxazole-Benzenesulfonamide Derivatives Inhibit HIV-1 Reverse Transcriptase Interaction with Cellular eEF1A and Reduce Viral Replication.

HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

Carbonic Anhydrase XII Inhibitors Overcome Temozolomide Resistance in Glioblastoma.

The natural product primary sulfonamide, psammaplin C (1), when used in combination with clinically used chemotherapeutic drugs, including temozolomide, reverses multidrug resistance and increases survival in glioblastoma, a highly aggressive primary brain tumor. We showed previously that the mechanism of action of 1 is novel, acting to indirectly interfere with P-glycoprotein drug efflux activity as a consequence of carbonic anhydrase XII (CA XII) inhibition. To build structure-activity relationships, 45 derivatives of 1 were designed, synthesized, and evaluated against a panel of CA isoforms. Compound 55 was identified as a potent inhibitor of CA XII ( Ki = 0.56 nM) and was investigated in vitro and in vivo using samples from glioblastoma patients. The results strengthen the possibility that co-therapy of temozolomide with a CA XII inhibitor may more effectively treat glioblastoma by suppressing an important temozolomide resistance mechanism.

Carbonic Anhydrase XII Inhibitors Overcome P-Glycoprotein-Mediated Resistance to Temozolomide in Glioblastoma.

The role of carbonic anhydrase XII (CAXII) in the chemoresistance of glioblastoma is unexplored. We found CAXII and P-glycoprotein (Pgp) coexpressed in neurospheres derived from 3 of 3 patients with different genetic backgrounds and low response to temozolomide (time to recurrence: 6-9 months). CAXII was necessary for the Pgp efflux of temozolomide and second-line chemotherapeutic drugs, determining chemoresistance in neurospheres. Psammaplin C, a potent inhibitor of CAXII, resensitized primary neurospheres to temozolomide by reducing temozolomide efflux via Pgp. This effect was independent of other known temozolomide resistance factors present in the patients. The overall survival in orthotopic patient-derived xenografts of temozolomide-resistant neurospheres, codosed with Psammaplin C and temozolomide, was significantly increased over temozolomide-treated (P < 0.05) and untreated animals (P < 0.02), without detectable signs of systemic toxicity. We propose that a CAXII inhibitor in combination with temozolomide may provide a new and effective approach to reverse chemoresistance in glioblastoma stem cells. This novel mechanism of action, via the interaction of CAXII and Pgp, ultimately blocks the efflux function of Pgp to improve glioblastoma patient outcomes.

Stereoselective Synthesis of Highly Functionalized Arabinosyl Nucleosides through Application of an N-Nitro Protecting Group.

2'-Deoxy-2',5-disubstituted arabinosyl uridine derivatives bearing a halogen (Cl, Br or I) at C2' and an ethynyl group at C5 have been synthesized in 6 steps from 2',3',5'-tri- O-acetyl-5-iodo-uridine in overall yields of 61% (compound 3, Cl), 47% (compound 4, Br), and 19% (compound 5, I). Stabilization of a 2'- O-triflyl leaving group intermediate to overcome spontaneous intramolecular 2,2'-anhydro uridine formation was pivotal to the synthesis. Specifically, to favor SN2 reaction with a halogen nucleophile over intramolecular cyclization, the nucleophilicity of O-2 oxygen was reduced by incorporation of an adjacent electron withdrawing nitro substituent at N-3. The introduction of the 3- N-nitro group proceeded rapidly (nitronium trifluoroacetate, 1 min) and in quantitative yield. A one-pot method to remove the 3- N-nitro group by reductive nitration (zinc metal in acetic acid, 5 min) and the silyl protecting groups of the alkyne and 3',5' hydroxyls (fluoride reagent, 16 h) was established as the final synthetic step. This application of the 3- N-nitro protecting group addresses the significant shortfalls of the conventional approach to synthesis of 2' modified nucleosides, wherein condensation of a 2' modified sugar fragment with a pyrimidine base provides poor stereocontrol of N-glycosylation, low yields and incompatibility with 2' iodo sugars.

Synthesis, structure and bioactivity of primary sulfamate-containing natural products.

Here we report the synthesis of natural products (NPs) 5'-O-sulfamoyl adenosine 1 and 5'-O-sulfamoyl-2-chloroadenosine 2. As primary sulfamates these compounds represent an uncommon class of NPs, furthermore there are few NPs known that contain a NS bond. Compounds 1 and 2 were evaluated for inhibition of carbonic anhydrases (CA), a metalloenzyme family where the primary sulfamate is known to coordinate to the active site zinc and form key hydrogen bonds with adjacent CA active site residues. Both NPs were good to moderate CA inhibitors, with compound 2 a 20-50-fold stronger CA inhibitor (Ki values 65-234 nM) than compound 1. The protein X-ray crystal structures of 1 and 2 in complex with CA II show that it is not the halogen-hydrophobic interactions that give compound 2 a greater binding energy but a slight movement in orientation of the ribose ring that allows better hydrogen bonds to CA residues. Compounds 1 and 2 were further investigated for antimicrobial activity against a panel of microbes relevant to human health, including Gram-negative bacteria (4 strains), Gram-positive bacteria (1 strain) and yeast (2 strains). Antimicrobial activity and selectivity was observed. The minimum inhibitory concentration (MIC) of NP 1 was 10 µM against Gram-positive Staphylococcus aureus and NP 2 was 5 µM against Gram-negative Escherichia coli. This is the first time that NP primary sulfamates have been assessed for inhibition and binding to CAs, with systematic antimicrobial activity studies also reported.

Identification of a New Zinc Binding Chemotype by Fragment Screening.

The discovery of a new zinc binding chemotype from screening a nonbiased fragment library is reported. Using the orthogonal fragment screening methods of native state mass spectrometry and surface plasmon resonance a 3-unsubstituted 2,4-oxazolidinedione fragment was found to have low micromolar binding affinity to the zinc metalloenzyme carbonic anhydrase II (CA II). This affinity approached that of fragment sized primary benzenesulfonamides, the classical zinc binding group found in most CA II inhibitors. Protein X-ray crystallography established that 3-unsubstituted 2,4-oxazolidinediones bound to CA II via an interaction of the acidic ring nitrogen with the CA II active site zinc, as well as two hydrogen bonds between the oxazolidinedione ring oxygen and the CA II protein backbone. Furthermore, 3-unsubstituted 2,4-oxazolidinediones appear to be a viable starting point for the development of an alternative class of CA inhibitor, wherein the medicinal chemistry pedigree of primary sulfonamides has dominated for several decades.

Screening the Medicines for Malaria Venture Pathogen Box across Multiple Pathogens Reclassifies Starting Points for Open-Source Drug Discovery.

Open-access drug discovery provides a substantial resource for diseases primarily affecting the poor and disadvantaged. The open-access Pathogen Box collection is comprised of compounds with demonstrated biological activity against specific pathogenic organisms. The supply of this resource by the Medicines for Malaria Venture has the potential to provide new chemical starting points for a number of tropical and neglected diseases, through repurposing of these compounds for use in drug discovery campaigns for these additional pathogens. We tested the Pathogen Box against kinetoplastid parasites and malaria life cycle stages in vitro Consequently, chemical starting points for malaria, human African trypanosomiasis, Chagas disease, and leishmaniasis drug discovery efforts have been identified. Inclusive of this in vitro biological evaluation, outcomes from extensive literature reviews and database searches are provided. This information encompasses commercial availability, literature reference citations, other aliases and ChEMBL number with associated biological activity, where available. The release of this new data for the Pathogen Box collection into the public domain will aid the open-source model of drug discovery. Importantly, this will provide novel chemical starting points for drug discovery and target identification in tropical disease research.

Synthesis of Novel Saccharin Derivatives.

The synthesis of saccharin (1,2-benzisothiazol-3-one-1,1-dioxide) derivatives substituted on the benzene ring has seen limited development despite the longevity of this compound's use as an artificial sweetener. This type of saccharin derivative would however present attractive properties for the development of new bioactive, drug-like small molecule compounds. Here we report the derivatisation of the benzene ring of saccharin using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) to synthesise a diverse library of novel saccharin-1,2,3-triazole conjugates. All library compounds retain the capability for interactions with biomolecules via the unmodified sulfonamide and lactam groups of the parent saccharin core heterocycle. The compounds also encompass alternate orientations of the 1,2,3-triazole heterocycle, thus further adding diversity to the potential hydrogen bonding interactions of these compounds with biomolecules of therapeutic interest. Our findings demonstrate that specifically functionalized derivatives of saccharin may be prepared from either saccharin azide or saccharin alkyne building blocks in high yield using CuAAC.