Methazolamide Reduces the AQP5 mRNA Expression and Immune Cell Migration-A New Potential Drug in Sepsis Therapy?

Sepsis is a life-threatening condition caused by the dysregulated host response to infection. Novel therapeutic options are urgently needed and aquaporin inhibitors could suffice as aquaporin 5 (Aqp5) knockdown provided enhanced sepsis survival in a murine sepsis model. Potential AQP5 inhibitors provide sulfonamides and their derivatives. In this study, we tested the hypothesis that sulfonamides reduce AQP5 expression in different conditions. The impact of sulfonamides on AQP5 expression and immune cell migration was examined in cell lines REH and RAW 264.7 by qPCR, Western blot and migration assay. Subsequently, whether furosemide and methazolamide are capable of reducing AQP5 expression after LPS incubation was investigated in whole blood samples of healthy volunteers. Incubation with methazolamide (10-5 M) and furosemide (10-6 M) reduced AQP5 mRNA and protein expression by about 30% in REH cells. Pre-incubation of the cells with methazolamide reduced cell migration towards SDF1-α compared to non-preincubated cells to control level. Pre-incubation with methazolamide in PBMCs led to a reduction in LPS-induced AQP5 expression compared to control levels, while furosemide failed to reduce it. Methazolamide appears to reduce AQP5 expression and migration of immune cells. However, after LPS administration, the reduction in AQP5 expression by methazolamide is no longer possible. Hence, our study indicates that methazolamide is capable of reducing AQP5 expression and has the potential to be used in sepsis prophylaxis.

Revealing mechanism of Methazolamide for treatment of ankylosing spondylitis based on network pharmacology and GSEA.

Methazolamide is a carbonic anhydrase (CA) inhibitor with satisfactory safety. Our previous studies have demonstrated the elevation of CA1 expression and the therapeutic effect of Methazolamide in Ankylosing spondylitis (AS). In this study, we explored the pathogenic role of CA1 and the pharmacological mechanism of Methazolamide in AS through Gene Set Enrichment Analysis (GSEA) and network pharmacology. Seven out of twelve CA1 related gene sets were enriched in AS group. CA1 was core enriched in above seven gene sets involving zinc ion binding, arylesterase activity and one carbon metabolic process. Functional analysis of the candidate target genes obtained from the intersection of AS associated genes and Methazolamide target genes indicated that Methazolamide exerts therapeutic effects on AS mainly through inflammatory pathways which regulate the production of tumor necrosis factor, IL-6 and nitric oxide. PTGS2, ESR1, GSK3ß, JAK2, NOS2 and CA1 were selected as therapeutic targets of Methazolamide in AS. Molecular docking and molecular dynamics simulations were performed successfully. In addition, we innovatively obtained the intersection of Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and GSEA results, and found that 18 GO terms and 5 KEGG terms were indicated in the pharmacological mechanism of Methazolamide in AS, involving bone mineralization, angiogenesis, inflammation, and chemokine signaling pathways. Nevertheless, validation for these mechanisms is needed in vivo/vitro experiments.

Managing primary open-angle glaucoma in the setting of suboptimal surgical outcomes in the fellow eye.

A 62-year-old woman with mild myopia presented to her local optometrist for a routine examination and was found to have intraocular pressure (IOP) of 30 mm Hg in both eyes and cupped nerves. She had a family history of glaucoma in her father. She was started on latanoprost in both eyes and was referred for a glaucoma evaluation. On initial evaluation, her IOP was 25 mm Hg in the right eye and 26 mm Hg in the left eye. Central corneal thickness measured 592 µm in the right eye and 581 µm in the left eye. Her angles were open to gonioscopy without any peripheral anterior synechia. She had 1+ nuclear sclerosis with a corrected distance visual acuity (CDVA) of 20/25 in the right eye and 20/30- in the left eye and uncorrected near visual acuity of J1+ in each eye. Her nerves were 0.85 mm in the right eye and 0.75 mm in the left eye. Optical coherence tomography (OCT) showed retinal nerve fiber layer thinning and a dense superior arcuate scotoma into fixation in her right eye, and superior and inferior arcuate scotomas in her left eye (Figures 1 and 2JOURNAL/jcrs/04.03/02158034-202307000-00019/figure1/v/2023-06-26T195222Z/r/image-tiffJOURNAL/jcrs/04.03/02158034-202307000-00019/figure2/v/2023-06-26T195222Z/r/image-tiff, Supplemental Figures 1 and 2, available at http://links.lww.com/JRS/A882 and http://links.lww.com/JRS/A883). She was successively trialed on fixed combination brimonidine-timolol, dorzolamide, and netarsudil, in addition to her latanoprost, but her IOP remained in the mid- to upper 20s in both eyes. The addition of acetazolamide lowered the pressure to 19 mm Hg in both eyes, but she tolerated it poorly. Methazolamide was also attempted with similar side effects. We elected to perform left eye cataract surgery combined with 360-degree viscocanaloplasty and insertion of a Hydrus microstent (Alcon Laboratories, Inc.). Surgery was uncomplicated with IOP of 16 mm Hg on postoperative day 1 with no glaucoma medications. However, by postoperative week 3, IOP returned to 27 mm Hg, and despite restarting latanoprost-netarsudil and finishing her steroid taper, IOP remained at 27 mm Hg by postoperative week 6. Brimonidine-timolol was added back to her left eye regimen and at postoperative week 8, IOP had elevated to 45 mm Hg. Maximizing her therapy with the addition of topical dorzolamide and oral methazolamide brought her IOP back down to 30 mm Hg. At that point, the decision was made to proceed with trabeculectomy of the left eye. The trabeculectomy was uneventful. However, postoperative attempts to augment filtration were rendered less successful by extremely thick Tenon layer. At her most recent follow-up the pressure in the left eye was mid-teens with brimonidine-timolol and dorzolamide. Her right eye IOP is in the upper 20s on maximum topical therapy. Knowing her postoperative course in the left eye, how would you manage the right eye? In addition to currently available options, would you consider a supraciliary shunt such as the MINIject (iSTAR) if such a device were U.S. Food and Drug Administration (FDA)-approved?

The Impact of Acetazolamide and Methazolamide on Exercise Performance in Normoxia and Hypoxia.

Doherty, Connor J., Jou-Chung Chang, Benjamin P. Thompson, Erik R. Swenson, Glen E. Foster, and Paolo B. Dominelli. The impact of acetazolamide and methazolamide on exercise performance in normoxia and hypoxia. High Alt Med Biol. 24:7-18, 2023.-Carbonic anhydrase (CA) inhibitors are commonly prescribed for acute mountain sickness (AMS). In this review, we sought to examine how two CA inhibitors, acetazolamide (AZ) and methazolamide (MZ), affect exercise performance in normoxia and hypoxia. First, we briefly describe the role of CA inhibition in facilitating the increase in ventilation and arterial oxygenation in preventing and treating AMS. Next, we detail how AZ affects exercise performance in normoxia and hypoxia and this is followed by a discussion on MZ. We emphasize that the overarching focus of the review is how the two drugs potentially affect exercise performance, rather than their ability to prevent/treat AMS per se, their interrelationship will be discussed. Overall, we suggest that AZ hinders exercise performance in normoxia, but may be beneficial in hypoxia. Based upon head-to-head studies of AZ and MZ in humans on diaphragmatic and locomotor strength in normoxia, MZ may be a better CA inhibitor when exercise performance is crucial at high altitude.

Sulphonamide inhibition studies of the ß-carbonic anhydrase GsaCAß present in the salmon platyhelminth parasite Gyrodactylus salaris.

A ß-class carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Monogenean platyhelminth Gyrodactylus salaris, a fish parasite, GsaCAß, has been investigated for its inhibitory effects with a panel of sulphonamides and sulfamates, some of which in clinical use. Several effective GsaCAß inhibitors were identified, belonging to simple heterocyclic sulphonamides, the deacetylated precursors of acetazolamide and methazolamide (KIsof 81.9-139.7 nM). Many other simple benezene sulphonamides and clinically used agents, such as acetazolamide, methazolamide, ethoxzolamide, dorzolamide, benzolamide, sulthiame and hydrochlorothiazide showed inhibition constants <1 µM. The least effective GsaCAß inhibitors were 4,6-disubstituted-1,3-benzene disulfonamides, with KIs in the range of 16.9-24.8 µM. Although no potent GsaCAß-selective inhibitors were detected so far, this preliminary investigation may be helpful for better understanding the inhibition profile of this parasite enzyme and for the potential development of more effective and eventually parasite-selective inhibitors.

Association of HLA-C*01:02 with methazolamide-induced toxic epidermal necrolysis.

We identified a Yi Chinese female patient from an ethnic minority group with methazolamide-induced toxic epidermal necrolysis. Genotyping revealed that she and her immediate family members carried the HLA-C*01:02 haplotypes, known to be associated with methazolamide-induced toxic epidermal necrolysis.

Detection, identification, characterization, and high-performance liquid chromatography quantification of five impurities from a methazolamide product.

Methazolamide is an important carbonic anhydrase inhibitor and is mainly used for the treatment of glaucoma. Studies are extremely rare regarding the impurities in methazolamide products. In this work, the high-performance liquid chromatography/high-performance liquid chromatography-mass spectrometry methods were established for the analysis of impurities in methazolamide products. Five impurities (A, B, C, D, and E) were detected using the established high-performance liquid chromatography/high-performance liquid chromatography-mass spectrometry methods. Of these impurities, impurities A, B, and D are known compounds, and impurities C and E are novel compounds that have never been reported before. The identities of impurities A, B, D, and E were recognized by comparing their retention times and mass spectra with those of synthesized standard compounds under the same high-performance liquid chromatography-mass spectrometry conditions. Moreover, the structures of impurities C and E were characterized using a variety of analytical techniques including multidimensional nuclear magnetic resonance spectroscopy, Fourier transforming infrared spectroscopy, ultraviolet-visible absorption spectroscopy, and high-resolution quadrupole time-of-flight mass spectrometry. All of the five impurities are structural analogs of methazolamide. The formation mechanisms of these impurities were discussed.

Unique motif shared by HLA-B*59:01 and HLA-B*55:02 is associated with methazolamide-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in Han Chinese.

BACKGROUND: Methazolamide (MTZ) has been occasionally linked to the lethal Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), which are associated with HLA-B*59:01. However, some MTZ-induced SJS/TEN (MTZ-SJS/TEN) cases are negative for HLA-B*59:01, implying that other genetic factors besides HLA-B*59:01 are contributing to MTZ-SJS/TEN. OBJECTIVES: To comprehensively identify HLA and non-HLA genetic susceptibility to MTZ-SJS/TEN in Han Chinese. METHODS: Eighteen patients with MTZ-SJS/TEN, 806 subjects of the population control and 74 MTZ-tolerant individuals were enrolled in this study. Both exome-wide and HLA-based association studies were conducted. Molecular docking analysis was employed to simulate the interactions between MTZ and risk HLA proteins. RESULTS: We found a strong signal in the major histocompatibility complex region on chromosome 6 with 22 SNPs reaching exome-wide significance. Compared with MTZ-tolerant controls, a significant association of HLA-B*59:01 with MTZ-SJS/TEN was validated [odds ratio (OR) = 146.00, 95% confidence interval (CI): 16.12-1321.98; P = 6.19 × 10-10 ]. Moreover, 66.7% of MTZ-SJS/TEN patients negative for HLA-B*59:01 were carriers of HLA-B*55:02, whilst 2.7% of the tolerant individuals were observed with HLA-B*55:02 (OR = 71.00, 95% CI: 7.84-643.10; P = 1.43 × 10-4 ). Within HLA-B protein, the E45-L116 motif could completely explain the association of HLA-B*59:01 and HLA-B*55:02 with MTZ-SJS/TEN (OR = 119.33, 95% CI: 29.19-1227.96; P = 4.36 × 10-13 ). Molecular docking analysis indicated that MTZ binds more stably to the pocket of HLA-B*59:01 and HLA-B*55:02 than to that of non-risk alleles of HLA-B*40:01 and HLA-C*01:02. CONCLUSIONS: This study confirmed the association of HLA-B*59:01 with MTZ-SJS/TEN and identified HLA-B*55:02 as a novel risk allele in Han Chinese with the largest sample size to date. Notably, the rs41562914(A)-rs12697944(A) haplotype, encoding E45-L116, is capable of serving as a powerful genetic predictor for MTZ-SJS/TEN with a sensitivity of 89% and specificity of 96%.

Cardioprotective effects of N-methylacetazolamide mediated by inhibition of L-type Ca2+ channel current.

Our objective was to examine the effects of N-methylacetazolamide (NMA), a non­carbonic anhydrase inhibitor, on ischemia-reperfusion injury. Isolated rat hearts were assigned to the following groups: 1) Non-ischemic control (NIC):110 min of perfusion and 2) Ischemic control (IC): 30 min of global ischemia and 60 min of reperfusion (R). Both groups were repeated in presence of NMA (5 µM), administered during the first 10 min of R. Infarct size (IS) was measured by TTC staining. Developed pressure (LVDP) and end-diastolic pressure (LVEDP) of the left ventricle were used to assess systolic and diastolic function, respectively. The content of P-Akt, P-PKCε, P-Drp1 and calcineurin Aß were measured. In cardiomyocytes the L-type Ca2+ current (ICaL) was recorded with the whole-cell configuration of patch-clamp technique. The addition of NMA to non-ischemic hearts decreased 15% the contractility. In ischemic hearts (IC group), NMA decreased IS (22 ± 2% vs 32 ± 2%, p < 0.05) and improved the post-ischemic recovery of myocardial function. At the end of R, LVDP was 54 ± 7% vs 18 ± 3% and LVEDP was 23 ± 8 vs. 55 ± 7 mmHg ¨p < 0.05¨. The level of P-Akt, P-PKCε and P-Drp1 increased and the expression of calcineurin Aß decreased in NMA treated hearts. Peak ICaL density recorded at 0 mV was smaller in myocytes treated with NMA than in non-treated cells (-1.91 ± 0.15 pA/pF vs -2.32 ± 0.17 pA/pF, p < 0.05). These data suggest that NMA protects the myocardium against ischemia-reperfusion injury through an attenuation of mitochondrial fission by calcineurin/Akt/PKCε-dependent pathways associated to the decrease of ICaL current.

Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry.

Coronavirus disease 2019 (COVID-19) represents a systemic disease that may cause severe metabolic complications in multiple tissues including liver, kidney, and cardiovascular system. However, the underlying mechanisms and optimal treatment remain elusive. Our study shows that impairment of ACE2 pathway is a key factor linking virus infection to its secondary metabolic sequelae. By using structure-based high-throughput virtual screening and connectivity map database, followed with experimental validations, we identify imatinib, methazolamide, and harpagoside as direct enzymatic activators of ACE2. Imatinib and methazolamide remarkably improve metabolic perturbations in vivo in an ACE2-dependent manner under the insulin-resistant state and SARS-CoV-2-infected state. Moreover, viral entry is directly inhibited by these three compounds due to allosteric inhibition of ACE2 binding to spike protein on SARS-CoV-2. Taken together, our study shows that enzymatic activation of ACE2 via imatinib, methazolamide, or harpagoside may be a conceptually new strategy to treat metabolic sequelae of COVID-19.

Methazolamide Attenuates the Development of Diabetic Cardiomyopathy by Promoting ß-Catenin Degradation in Type 1 Diabetic Mice.

Methazolamide (MTZ), a carbonic anhydrase inhibitor, has been shown to inhibit cardiomyocyte hypertrophy and exert a hypoglycemic effect in patients with type 2 diabetes and diabetic db/db mice. However, whether MTZ has a cardioprotective effect in the setting of diabetic cardiomyopathy is not clear. We investigated the effects of MTZ in a mouse model of streptozotocin-induced type 1 diabetes mellitus (T1DM). Diabetic mice received MTZ by intragastric gavage (10, 25, or 50 mg/kg, daily for 16 weeks). In the diabetic group, MTZ significantly reduced both random and fasting blood glucose levels and improved glucose tolerance in a dose-dependent manner. MTZ ameliorated T1DM-induced changes in cardiac morphology and dysfunction. Mechanistic analysis revealed that MTZ blunted T1DM-induced enhanced expression of ß-catenin. Similar results were observed in neonatal rat cardiomyocytes (NRCMs) and adult mouse cardiomyocytes treated with high glucose or Wnt3a (a ß-catenin activator). There was no significant change in ß-catenin mRNA levels in cardiac tissues or NRCMs. MTZ-mediated ß-catenin downregulation was recovered by MG132, a proteasome inhibitor. Immunoprecipitation and immunofluorescence analyses showed augmentation of AXIN1-ß-catenin interaction by MTZ in T1DM hearts and in NRCMs treated with Wnt3a; thus, MTZ may potentiate AXIN1-ß-catenin linkage to increase ß-catenin degradation. Overall, MTZ may alleviate cardiac hypertrophy by mediating AXIN1-ß-catenin interaction to promote degradation and inhibition of ß-catenin activity. These findings may help inform novel therapeutic strategy to prevent heart failure in patients with diabetes.

Gamma sterilization and in vivo evaluation of cationic nanostructured lipid carriers as potential ocular delivery systems for antiglaucoma drugs.

Solid lipid nanoparticles and nanostructured lipid carriers showed promising results for enhancement of ocular bioavailability of drugs with poor corneal permeability. One of these drugs is methazolamide, which is an orally administered carbonic anhydrase inhibitor for glaucoma treatment. However, sterilization by autoclaving may result in loss of the physical properties of lipid nanoparticles such as particle size and surface charge. Here, we evaluated gamma radiation as an alternative sterilization method. Methazolamide loaded nanostructured lipid carriers were optimized using 23 factorial design. Optimized formulations contained 6% lipid (85% solid lipid (Cetostearyl alcohol and glyceryl behenate) and 15% oil either medium chain triglycerides or isopropyl myristate) stabilized by 2% polysorbate 80 and 0.15% stearylamine. Nanoparticles were cationic, smaller than 500 nm, and had an entrapment efficiency of about 30%. They released methazolamide within 8 hours and showed a 5-fold enhanced reduction in intraocular pressure compared to methazolamide solution. Gamma sterilization was superior to autoclaving in preserving entrapped methazolamide, size, and surface charge of lipid nanoparticles. These findings demonstrate that gamma radiation is a viable alternative to autoclaving for sterilizing lipid nanoparticles. Moreover, this proves that nanostructured lipid carriers enhance pharmacological response of topically administered methazolamide for treating glaucoma.

Combined methazolamide and theophylline improves oxygen saturation but not exercise performance or altitude illness in acute hypobaric hypoxia.

NEW FINDINGS: What is the central question of this study? Does the combination of methazolamide and theophylline reduce symptoms of acute mountain sickness (AMS) and improve aerobic performance in acute hypobaric hypoxia? What is the main finding and its importance? The oral combination of methazolamide (100 BID) and theophylline (300 BID) improved arterial oxygen saturation but did not reduce symptoms of AMS and impaired aerobic performance. We do not recommend this combination of drugs for prophylaxis against the acute negative effects of hypobaric hypoxia. ABSTRACT: A limited number of small studies have suggested that methazolamide and theophylline can independently reduce symptoms of acute mountain sickness (AMS) and, if taken together, can improve aerobic exercise performance in normobaric hypoxia. We performed a randomized, double-blind, placebo-controlled, cross-over study to determine if the combination of oral methazolamide and theophylline could provide prophylaxis against AMS and improve aerobic performance in hypobaric hypoxia (∼4875 m). Volunteers with histories of AMS were screened at low altitude (1650 m) and started combined methazolamide (100 mg BID) and theophylline (300 mg BID) treatment, or placebo, 72 h prior to decompression. Baseline AMS (Lake Louise Questionnaire), blood (haemoglobin, haematocrit), cognitive function, ventilatory and pulse oximetry ( SpO2 ) measures were assessed at low altitude and repeated between 4 and 10 h of exposure to hypobaric hypoxia (PB  = 425 mmHg). Aerobic exercise performance was assessed during a 12.5 km cycling time trial (TT) after 4 h of hypobaric hypoxia. Subjects repeated all experimental procedures after a 3-week washout period. Differences between drug and placebo trials were evaluated using repeated measures ANOVA (α = 0.05). The drugs improved resting SpO2 by ∼4% (P < 0.01), but did not affect the incidence or severity of AMS or cognitive function scores relative to placebo. Subjects' performance on the 12.5 km TT was ∼3% worse when taking the drugs (P < 0.01). The combination of methazolamide and theophylline in the prescribed dosages is not recommended for use at high altitude as it appears to have no measurable effect on AMS and can impair aerobic performance.

Whole blood or plasma: what is the ideal matrix for pharmacokinetic-driven drug candidate selection?

In the present era of drug development, quantification of drug concentrations following pharmacokinetic studies has preferentially been performed using plasma as a matrix rather than whole blood. However, it is critical to realize the difference between measuring drug concentrations in blood versus plasma and the consequences thereof. Pharmacokinetics using plasma data may be misleading if concentrations differ between plasma and red blood cells (RBCs) because of differential binding in blood. In this review, factors modulating the partitioning of drugs into RBCs are discussed and the importance of determining RBC uptake of drugs for drug candidate selection is explored. In summary, the choice of matrix (plasma vs whole blood) is an important consideration to be factored in during drug discovery.

Real-Time Quantitative In-Cell NMR: Ligand Binding and Protein Oxidation Monitored in Human Cells Using Multivariate Curve Resolution.

In-cell NMR can investigate protein conformational changes at atomic resolution, such as those changes induced by drug binding or chemical modifications, directly in living human cells, and therefore has great potential in the context of drug development as it can provide an early assessment of drug potency. NMR bioreactors can greatly improve the cell sample stability over time and, more importantly, allow for recording in-cell NMR data in real time to monitor the evolution of intracellular processes, thus providing unique insights into the kinetics of drug-target interactions. However, current implementations are limited by low cell viability at >24 h times, the reduced sensitivity compared to "static" experiments and the lack of protocols for automated and quantitative analysis of large amounts of data. Here, we report an improved bioreactor design which maintains human cells alive and metabolically active for up to 72 h, and a semiautomated workflow for quantitative analysis of real-time in-cell NMR data relying on Multivariate Curve Resolution. We apply this setup to monitor protein-ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing further analysis by fitting with kinetic models. This unique approach can therefore be applied to investigate complex kinetic behaviors of macromolecules in a cellular setting, and could be extended in principle to any real-time NMR application in live cells.

Methazolamide in high-altitude illnesses.

As a carbonic anhydrase inhibitor and a methylated lipophilic analogue of acetazolamide, Methazolamide has higher lipid solubility, less plasma protein binding and renal excretion, and fewer side effects, compared to acetazolamide. Methazolamide can increase systemic metabolic acidosis and sequentially improve ventilation and oxygenation level. The increased oxygenation level leads to reduced reactive oxygen species (ROS) production, relived cerebral edema, mitigated hypoxic pulmonary vasoconstriction, abrogated hypoxic fatigue, and decreased excessive erythrocytosis. In addition to the effect as a carbonic anhydrase inhibitor, methazolamide directly activates the transcription factor anti-oxidative nuclear factor-related factor 2 (Nrf2) and inhibits interleukin-1ß (IL-1ß) release. These pharmacological functions of methazolamide are beneficial for the prevention and treatment of high-altitude illnesses. Besides, methazolamide causes less fatigue side effects than acetazolamide does. It is also worth noting that several studies suggested that a lower dose of methazolamide has similar prophylaxis and treatment efficacy in acute mountain sickness (AMS) to a higher dose of acetazolamide. Given methazolamide's advantages over acetazolamide, methazolamide may thus represent an alternative for acetazolamide when taken for high-altitude illnesses prophylaxis and treatment. However, more in-depth clinical trials are needed to fully evaluate this efficacy of methazolamide.

Sulfonamide Inhibition Studies of the ß-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora.

A new ß-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: kcat of (7.9 ± 0.2) × 105 s-1, and kcat/Km of (9.5 ± 0.12) × 107 M-1∙s-1. An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with KIs in the range of 54-95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.