Melissa A. Pinard

A class of sulfonamide carbonic anhydrase inhibitors with neuropathic pain modulating effects

A series of benzene sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors which incorporate lipophilic 4-alkoxy- and 4-aryloxy moieties, together with several derivatives of ethoxzolamide and sulfanilamide are reported. These derivatives were investigated as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) of which multiple isoforms are known, and some appear to be involved in pain. These sulfonamides showed modest inhibition against the cytosolic isoform CA I, but were generally effective, low nanomolar CA II, VII, IX and XII inhibitors. X-ray crystallographic data for the adduct of several such sulfonamides with CA II allowed us to rationalize the good inhibition data. In a mice model of neuropathic pain induced by oxaliplatin, one of the strong CA II/VII inhibitors reported here induced a long lasting pain relieving effect, a fact never observed earlier. This is the first report of rationally designed sulfonamide CA inhibitors with pain effective modulating effects.

Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases.

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. Their pivotal role in metabolism, ubiquitous nature, and multiple isoforms (CA I-XIV) has made CAs an attractive drug target in clinical applications. The usefulness of CA inhibitors (CAIs) in the treatment of glaucoma and epilepsy are well documented. In addition several isoforms of CAs (namely, CA IX) also serve as biological markers for certain tumors, and therefore they have the potential for useful applications in the treatment of cancer. This is a structural study on the binding interactions of the widely used CA inhibitory drugs brinzolamide (marketed as Azopt®) and dorzolamide (marketed as Trusopt®) with CA II and a CA IX-mimic, which was created via site-directed mutagenesis of CA II cDNA such that the active site resembles that of CA IX. Also the inhibition of CA II and CA IX and molecular docking reveal brinzolamide to be a more potent inhibitor among the other catalytically active CA isoforms compared to dorzolamide. The structures show that the tail end of the sulfonamide inhibitor is critical in forming stabilizing interactions that influence tight binding; therefore, for future drug design it is the tail moiety that will ultimately determine isoform specificity.

Targeting Carbonic Anhydrase IX Activity and Expression

Metastatic tumors are often hypoxic exhibiting a decrease in extracellular pH (~6.5) due to a metabolic transition described by the Warburg Effect. This shift in tumor cell metabolism alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments; hence hindering treatment of aggressive cancers. As a result, tumors exhibiting this phenotype are directly associated with poor prognosis and decreased survival rates in cancer patients. A key component to this tumor microenvironment is carbonic anhydrase IX (CA IX). Knockdown of CA IX expression or inhibition of its activity has been shown to reduce primary tumor growth, tumor proliferation, and also decrease tumor resistance to conventional anti-cancer therapies. As such several approaches have been taken to target CA IX in tumors via small-molecule, anti-body, and RNAi delivery systems. Here we will review recent developments that have exploited these approaches and provide our thoughts for future directions of CA IX targeting for the treatment of cancer.

Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3 −. Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors.

A Class of 4-Sulfamoylphenyl-ω-aminoalkyl Ethers with Effective Carbonic Anhydrase Inhibitory Action and Antiglaucoma Effects

We report a series of 4-sulfamoylphenyl-ω-aminoalkyl ethers as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The structure–activity relationship was drawn for the inhibition of four physiologically relevant isoforms: hCA I, II, IX, and XII. Many of these compounds were highly effective, low nanomolar inhibitors of all CA isoforms, whereas several isoform-selective were also identified. X-ray crystal structures of two new sulfonamides bound to the physiologically dominant CA II isoform showed the tails of these derivatives bound within the hydrophobic half of the enzyme active site through van der Waals contacts with Val135, Leu198, Leu204, Trp209, Pro201, and Pro202 amino acids. One of the highly water-soluble compound (as trifluoroacetate salt) showed effective IOP lowering properties in an animal model of glaucoma. Several fluorescent sulfonamides incorporating either the fluorescein-thiourea (7a–c) or tetramethylrhodamine-thiourea (9a,b) moieties were also obtained and showed interesting CA inhibitory properties for the tumor-associated isoforms CA IX and XII.

Structure and inhibition studies of a type II beta-carbonic anhydrase psCA3 from Pseudomonas aeruginosa.

Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The β-class CAs (β-CAs) are expressed in prokaryotes, fungi, plants, and more recently have been isolated in some animals. The β-CA class is divided into two subclasses, termed type I and II, defined by pH catalytic activity profile and active site structural configuration. Type I β-CAs display catalytic activity over a broad pH range (6.5-9.0) with the active site zinc tetrahedrally coordinated by three amino acids and a hydroxide/water. In contrast, type II β-CAs are catalytically active only at a pH 8 and higher where they adopt a functional active site configuration like that of type I. However, below pH 8 they are conformationally self-inactivated by the addition of a fourth amino acid coordinating the zinc and thereby displacing the zinc bound solvent. We have determined the structure of psCA3, a type II β-CA, isolated from Pseudomonas aeruginosa (P. aeruginosa) PAO1 at pH 8.3, in its open active state to a resolution of 1.9 Å. The active site zinc is coordinated by Cys42, His98, Cys101 and a water/hydroxide molecule. P. aeruginosa is a multi-drug resistant bacterium and displays intrinsic resistance to most of the currently used antibiotics; therefore, there is a need for new antibacterial targets. Kinetic data confirm that psCA3 belongs to the type II subclass and that sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. In vivo studies identified that among six tested inhibitors representing sulfonamides, inorganic anions, and small molecules, acetazolamide has the most significant dose-dependent inhibitory effect on P. aeruginosa growth.

The malignant brain tumor (MBT) domain protein SFMBT1 is an integral histone reader subunit of the LSD1 demethylase complex for chromatin association and epithelial-to-mesenchymal transition.

Background: The LSD1 histone demethylase regulates gene expression by demethylating chromatin. Results: SFMBT1 associates with the LSD1 complex and is essential for its chromatin association, histone demethylation, and induction of EMT. Conclusion: SFMBT1 is a novel, integral component of the LSD1 complex. Significance: Understanding how the LSD1 complex is recruited to chromatin may offer new opportunities for therapeutic intervention. Chromatin readers decipher the functional readouts of histone modifications by recruiting specific effector complexes for subsequent epigenetic reprogramming. The LSD1 (also known as KDM1A) histone demethylase complex modifies chromatin and represses transcription in part by catalyzing demethylation of dimethylated histone H3 lysine 4 (H3K4me2), a mark for active transcription. However, none of its currently known subunits recognizes methylated histones. The Snai1 family transcription factors are central drivers of epithelial-to-mesenchymal transition (EMT) by which epithelial cells acquire enhanced invasiveness. Snai1-mediated transcriptional repression of epithelial genes depends on its recruitment of the LSD1 complex and ensuing demethylation of H3K4me2 at its target genes. Through biochemical purification, we identified the MBT domain-containing protein SFMBT1 as a novel component of the LSD1 complex associated with Snai1. Unlike other mammalian MBT domain proteins characterized to date that selectively recognize mono- and dimethylated lysines, SFMBT1 binds di- and trimethyl H3K4, both of which are enriched at active promoters. We show that SFMBT1 is essential for Snai1-dependent recruitment of LSD1 to chromatin, demethylation of H3K4me2, transcriptional repression of epithelial markers, and induction of EMT by TGFβ. Carcinogenic metal nickel is a widespread environmental and occupational pollutant. Nickel alters gene expression and induces EMT. We demonstrate the nickel-initiated effects are dependent on LSD1-SFMBT1-mediated chromatin modification. Furthermore, in human cancer, expression of SFMBT1 is associated with mesenchymal markers and unfavorable prognosis. These results highlight a critical role of SFMBT1 in epigenetic regulation, EMT, and cancer.

Catalytic Mechanism of α-Class Carbonic Anhydrases: CO2 Hydration and Proton Transfer

The carbonic anhydrases (CAs; EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide (CO2) and dehydration of bicarbonate (HCO3 (-)) in a two-step ping-pong mechanism: [Formula: see text] CAs are ubiquitous enzymes and are categorized into five distinct classes (α, β, γ, δ and ζ). The α-class is found primarily in vertebrates (and the only class of CA in mammals), β is observed in higher plants and some prokaryotes, γ is present only in archaebacteria whereas the δ and ζ classes have only been observed in diatoms.The focus of this chapter is on α-CAs as the structure-function relationship is best understood for this class, in particular for humans. The reader is referred to other reviews for an overview of the structure and catalytic mechanism of the other CA classes. The overall catalytic site structure and geometry of α-CAs are described in the first section of this chapter followed by the kinetic studies, binding of CO2, and the proton shuttle network.

Reactive Center Loop (RCL) Peptides Derived from Serpins Display Independent Coagulation and Immune Modulating Activities

Serpins regulate coagulation and inflammation, binding serine proteases in suicide-inhibitory complexes. Target proteases cleave the serpin reactive center loop scissile P1–P1′ bond, resulting in serpin-protease suicide-inhibitory complexes. This inhibition requires a near full-length serpin sequence. Myxomavirus Serp-1 inhibits thrombolytic and thrombotic proteases, whereas mammalian neuroserpin (NSP) inhibits only thrombolytic proteases. Both serpins markedly reduce arterial inflammation and plaque in rodent models after single dose infusion. In contrast, Serp-1 but not NSP improves survival in a lethal murine gammaherpesvirus68 (MHV68) infection in interferon γ-receptor-deficient mice (IFNγR−/−). Serp-1 has also been successfully tested in a Phase 2a clinical trial. We postulated that proteolytic cleavage of the reactive center loop produces active peptide derivatives with expanded function. Eight peptides encompassing predicted protease cleavage sites for Serp-1 and NSP were synthesized and tested for inhibitory function in vitro and in vivo. In engrafted aorta, selected peptides containing Arg or Arg-Asn, not Arg-Met, with a 0 or +1 charge, significantly reduced plaque. Conversely, S-6 a hydrophobic peptide of NSP, lacking Arg or Arg-Asn with −4 charge, induced early thrombosis and mortality. S-1 and S-6 also significantly reduced CD11b+ monocyte counts in mouse splenocytes. S-1 peptide had increased efficacy in plasminogen activator inhibitor-1 serpin-deficient transplants. Plaque reduction correlated with mononuclear cell activation. In a separate study, Serp-1 peptide S-7 improved survival in the MHV68 vasculitis model, whereas an inverse S-7 peptide was inactive. Reactive center peptides derived from Serp-1 and NSP with suitable charge and hydrophobicity have the potential to extend immunomodulatory functions of serpins.

A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX

Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-), thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an Rcryst of 18.0% and an Rfree of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.