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1.
ChemMedChem ; 13(19): 2024-2029, 2018 10 08.
Article in English | MEDLINE | ID: mdl-30088334

ABSTRACT

Pseudomonas aeruginosa is a Gram-negative facultative anaerobe belonging to the Pseudomonadaceae family. It is a multidrug-resistant opportunistic human pathogen, a common cause of life-threatening nosocomial infections, and a key bacterial agent in cystic fibrosis and endocarditis. The bacterium exhibits intrinsic resistance to most antibacterial agents, including aminoglycosides and quinolones. Hence, the identification of new drug targets for P. aeruginosa is ongoing. PsCA3 is a ß-class carbonic anhydrase (ß-CA) that catalyzes the reversible hydration of carbon dioxide to bicarbonate and represents a new class of antimicrobial target. Previously, inhibitor screening studies of psCA3 have shown that a series of small anions including sulfamide (SFN), imidazole (IMD), and 4-methylimidazole (4MI), and thiocyanate (SCN) inhibit the enzyme with efficiencies in the micro- to millimolar range. Herein the X-ray crystal structures of these inhibitors in complex with psCA3 are presented and compared with human CA II. This structural survey into the binding modes of small anions forms the foundation for the development of inhibitors against ß-CAs and more selective inhibitors against P. aeruginosa.


Subject(s)
Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Carbonic Anhydrase III/metabolism , Carbonic Anhydrase Inhibitors/metabolism , Pseudomonas aeruginosa/enzymology , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Carbonic Anhydrase II/chemistry , Carbonic Anhydrase II/metabolism , Carbonic Anhydrase III/chemistry , Carbonic Anhydrase III/isolation & purification , Carbonic Anhydrase Inhibitors/chemistry , Catalytic Domain , Crystallography, X-Ray , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Kinetics , Protein Binding , Sulfonamides/chemistry , Sulfonamides/metabolism , Thiocyanates/chemistry , Thiocyanates/metabolism
2.
Biochemistry ; 57(7): 1096-1107, 2018 02 20.
Article in English | MEDLINE | ID: mdl-29227673

ABSTRACT

The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.


Subject(s)
Immunologic Factors/chemistry , Myxoma virus/chemistry , Peptides/chemistry , Serpins/chemistry , Viral Proteins/chemistry , Animals , CHO Cells , Cricetulus , Crystallography, X-Ray , Humans , Immunologic Factors/pharmacology , Mice , Mice, Inbred C57BL , Models, Molecular , Peptides/pharmacology , Poxviridae Infections/virology , Protein Conformation , Rabbits , Serpins/pharmacology , Viral Proteins/pharmacology
3.
J Virol ; 91(2)2017 Jan 15.
Article in English | MEDLINE | ID: mdl-27847360

ABSTRACT

Parvovirus capsids are small but complex molecular machines responsible for undertaking many of the steps of cell infection, genome packing, and cell-to-cell as well as host-to-host transfer. The details of parvovirus infection of cells are still not fully understood, but the processes must involve small changes in the capsid structure that allow the endocytosed virus to escape from the endosome, pass through the cell cytoplasm, and deliver the single-stranded DNA (ssDNA) genome to the nucleus, where viral replication occurs. Here, we examine capsid substitutions that eliminate canine parvovirus (CPV) infectivity and identify how those mutations changed the capsid structure or altered interactions with the infectious pathway. Amino acid substitutions on the exterior surface of the capsid (Gly299Lys/Ala300Lys) altered the binding of the capsid to transferrin receptor type 1 (TfR), particularly during virus dissociation from the receptor, but still allowed efficient entry into both feline and canine cells without successful infection. These substitutions likely control specific capsid structural changes resulting from TfR binding required for infection. A second set of changes on the interior surface of the capsid reduced viral infectivity by >100-fold and included two cysteine residues and neighboring residues. One of these substitutions, Cys270Ser, modulates a VP2 cleavage event found in ∼10% of the capsid proteins that also was shown to alter capsid stability. A neighboring substitution, Pro272Lys, significantly reduced capsid assembly, while a Cys273Ser change appeared to alter capsid transport from the nucleus. These mutants reveal additional structural details that explain cell infection processes of parvovirus capsids. IMPORTANCE: Parvoviruses are commonly found in both vertebrate and invertebrate animals and cause widespread disease. They are also being developed as oncolytic therapeutics and as gene therapy vectors. Most functions involved in infection or transduction are mediated by the viral capsid, but the structure-function correlates of the capsids and their constituent proteins are still incompletely understood, especially in relation to identifying capsid processes responsible for infection and release from the cell. Here, we characterize the functional effects of capsid protein mutations that result in the loss of virus infectivity, giving a better understanding of the portions of the capsid that mediate essential steps in successful infection pathways and how they contribute to viral infectivity.


Subject(s)
Capsid Proteins/chemistry , Capsid Proteins/metabolism , Parvoviridae Infections/virology , Parvovirus/physiology , Protein Conformation , Amino Acid Sequence , Capsid Proteins/genetics , Endopeptidases/metabolism , Host-Pathogen Interactions , Models, Molecular , Mutation , Protein Transport , Proteolysis , Receptors, Virus/metabolism , Structure-Activity Relationship , Virus Attachment
4.
Acta Crystallogr F Struct Biol Commun ; 72(Pt 7): 573-7, 2016 07.
Article in English | MEDLINE | ID: mdl-27380376

ABSTRACT

Cryoannealing has been demonstrated to improve the diffraction quality and resolution of crystals of the ß-carbonic anhydrase psCA3 concomitant with a change in space group. After initial flash-cooling in a liquid-nitrogen cryostream an X-ray diffraction data set from a psCA3 crystal was indexed in space group P21212 and was scaled to 2.6 Šresolution, but subsequent cryoannealing studies revealed induced protein rearrangements in the crystal contacts, which transformed the space group to I222, with a corresponding improvement of 0.7 Šin resolution. Although the change in diffraction resolution was significant, only minor changes in the psCA3 structure, which retained its catalytic `open' conformation, were observed. These findings demonstrate that cryoannealing can be successfully utilized to induce higher diffraction-quality crystals while maintaining enzymatically relevant conformations and may be useful as an experimental tool for structural studies of other enzymes where the initial diffraction quality is poor.


Subject(s)
Bacterial Proteins/chemistry , Carbonic Anhydrase III/chemistry , Pseudomonas aeruginosa/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbonic Anhydrase III/genetics , Carbonic Anhydrase III/metabolism , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Models, Molecular , Plasmids/chemistry , Plasmids/metabolism , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Secondary , Pseudomonas aeruginosa/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
5.
J Biol Chem ; 291(6): 2874-87, 2016 Feb 05.
Article in English | MEDLINE | ID: mdl-26620556

ABSTRACT

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.


Subject(s)
Blood Coagulation/drug effects , Herpesviridae Infections/immunology , Immunologic Factors , Membrane Proteins , Peptides , Rhadinovirus/immunology , Vasculitis/immunology , Animals , Blood Coagulation/immunology , Disease Models, Animal , Herpesviridae Infections/drug therapy , Humans , Immunologic Factors/chemical synthesis , Immunologic Factors/chemistry , Immunologic Factors/immunology , Jurkat Cells , Membrane Proteins/chemical synthesis , Membrane Proteins/chemistry , Membrane Proteins/pharmacology , Mice , Mice, Knockout , Peptides/chemical synthesis , Peptides/chemistry , Peptides/pharmacology , Vasculitis/drug therapy
6.
Acta Crystallogr F Struct Biol Commun ; 71(Pt 10): 1352-8, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26457530

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/metabolism , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Neoplasms/enzymology , Sucrose/metabolism , Binding Sites , Carbonic Anhydrase IX , Crystallization , Crystallography, X-Ray , Fluorometry , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics
7.
Biochemistry ; 54(43): 6631-8, 2015 Nov 03.
Article in English | MEDLINE | ID: mdl-26457866

ABSTRACT

Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/dehydration of CO2/HCO3(-) with rates approaching diffusion-controlled limits (kcat/KM ∼ 10(8) M(-1) s(-1)). This family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a ß-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryo-cooled crystallography. The structure has been refined to 1.6 Å resolution with R(cryst) and R(free) values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2 captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2 binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2 binding site was located at the dimer interface of psCA3. Interestingly, all ß-CAs function as dimers or higher-order oligomeric states, and the CO2 bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Carbon Dioxide/metabolism , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/metabolism , Pseudomonas aeruginosa/enzymology , Bacterial Proteins/genetics , Carbonic Anhydrase II/chemistry , Carbonic Anhydrase II/metabolism , Carbonic Anhydrases/genetics , Catalytic Domain , Crystallography, X-Ray , Humans , Ligands , Models, Molecular , Protein Conformation , Protein Structure, Quaternary , Pseudomonas aeruginosa/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Species Specificity
8.
Protein Sci ; 24(11): 1800-7, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26266677

ABSTRACT

Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 Å resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (∼ 4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems.


Subject(s)
Carbonic Anhydrase II/chemistry , Carbonic Anhydrase II/metabolism , Lysine/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Acetylation , Carbonic Anhydrase II/genetics , Escherichia coli/genetics , Humans , Lysine/chemistry , Models, Molecular , Protein Processing, Post-Translational , Protein Stability , Recombinant Proteins/genetics
9.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 8): 1745-56, 2015 Aug.
Article in English | MEDLINE | ID: mdl-26249355

ABSTRACT

Biocatalytic CO2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°C versus 59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO2 sequestration.


Subject(s)
Carbon Dioxide/metabolism , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/metabolism , Gammaproteobacteria/chemistry , Gammaproteobacteria/enzymology , Biocatalysis , Carbonic Anhydrases/genetics , Catalytic Domain , Crystallography, X-Ray , Enzyme Stability , Gammaproteobacteria/genetics , Humans , Models, Molecular , Protein Conformation , Protein Engineering , Protein Multimerization , Temperature
10.
J Med Chem ; 58(16): 6630-8, 2015 Aug 27.
Article in English | MEDLINE | ID: mdl-26203869

ABSTRACT

Inhibition of human carbonic anhydrase IX (hCA IX) has shown to be therapeutically advantageous for treating many types of highly aggressive cancers. However, designing selective inhibitors for hCA IX has been difficult due to its high structural homology and sequence similarity with off-target hCAs. Recently, the use of glucosyl sulfamate inhibitors has shown promise as selective inhibitors for hCA IX. In this study, we present five X-ray crystal structures, determined to a resolution of 1.7 Å or better, of both hCA II (a ubiquitous CA) and an engineered hCA IX-mimic in complex with selected glucosyl sulfamates and structurally rationalize mechanisms for hCA IX selectivity. Results from this study have allowed us, for the first time, to empirically "map" key interactions of the hCA IX active site in order to establish parameters needed to design novel hCA IX selective inhibitors.


Subject(s)
Antigens, Neoplasm/drug effects , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/drug effects , Sulfonic Acids/chemical synthesis , Sulfonic Acids/pharmacology , Carbonic Anhydrase IX , Humans , Isoenzymes/antagonists & inhibitors , Models, Molecular , Molecular Conformation , Structure-Activity Relationship , Substrate Specificity , X-Ray Diffraction
11.
Bioorg Med Chem ; 23(15): 4831-4838, 2015 Aug 01.
Article in English | MEDLINE | ID: mdl-26068018

ABSTRACT

Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The ß-class CAs (ß-CAs) are expressed in prokaryotes, fungi, plants, and more recently have been isolated in some animals. The ß-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 ß-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 ß-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 ß-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.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Carbonic Anhydrase II/antagonists & inhibitors , Carbonic Anhydrase Inhibitors/chemistry , Pseudomonas aeruginosa/enzymology , Bacterial Proteins/metabolism , Binding Sites , Boronic Acids/chemistry , Carbonic Anhydrase II/genetics , Carbonic Anhydrase II/metabolism , Carbonic Anhydrase Inhibitors/metabolism , Catalytic Domain , Crystallography, X-Ray , Dimerization , Drug Resistance, Multiple, Bacterial , Hydrogen-Ion Concentration , Kinetics , Molecular Dynamics Simulation , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sulfonamides/chemistry , Sulfonamides/metabolism , Sulfonic Acids/chemistry
12.
Bioorg Med Chem Lett ; 25(21): 4937-4940, 2015 Nov 01.
Article in English | MEDLINE | ID: mdl-25998503

ABSTRACT

Thiomicrospira crunogena XCL-2 expresses an α-carbonic anhydrase (TcruCA). Sequence alignments reveal that TcruCA displays a high sequence identity (>30%) relative to other α-CAs. This includes three conserved histidines that coordinate the active site zinc, a histidine proton shuttling residue, and opposing hydrophilic and hydrophobic sides that line the active site. The catalytic efficiency of TcruCA is considered moderate relative to other α-CAs (k(cat)/K(M)=1.1×10(7) M(-1) s(-1)), being a factor of ten less efficient than the most active α-CAs. TcruCA is also inhibited by anions with Cl(-), Br(-), and I(-), all showing Ki values in the millimolar range (53-361 mM). Hydrogen sulfide (HS(-)) revealed the highest affinity for TcruCA with a Ki of 1.1 µM. It is predicted that inhibition of TcruCA by HS(-) (an anion commonly found in the environment where Thiomicrospira crunogena is located) is a way for Thiomicrospira crunogena to regulate its carbon-concentrating mechanism (CCM) and thus the organism's metabolic functions. Results from this study provide preliminary insights into the role of TcruCA in the general metabolism of Thiomicrospira crunogena.


Subject(s)
Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Gammaproteobacteria/enzymology , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/chemistry , Dose-Response Relationship, Drug , Molecular Structure , Structure-Activity Relationship
13.
Biomed Res Int ; 2015: 453543, 2015.
Article in English | MEDLINE | ID: mdl-25811028

ABSTRACT

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.


Subject(s)
Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Drug Design , Biocatalysis/drug effects , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/chemistry , Catalytic Domain , Chelating Agents , Conserved Sequence , Humans , Hydrophobic and Hydrophilic Interactions , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Models, Molecular , Phylogeny , Protein Binding/drug effects , Solvents , Sulfonamides/chemistry
14.
Bioorg Med Chem ; 23(8): 1828-40, 2015 Apr 15.
Article in English | MEDLINE | ID: mdl-25766630

ABSTRACT

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.


Subject(s)
Analgesics/chemistry , Analgesics/therapeutic use , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/therapeutic use , Neuralgia/drug therapy , Sulfonamides/chemistry , Sulfonamides/therapeutic use , Animals , Carbonic Anhydrases/metabolism , Crystallography, X-Ray , Humans , Mice , Models, Molecular , Neuralgia/metabolism
15.
Molecules ; 20(2): 2323-48, 2015 Jan 30.
Article in English | MEDLINE | ID: mdl-25647573

ABSTRACT

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.


Subject(s)
Antigens, Neoplasm/physiology , Antineoplastic Agents/pharmacology , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/physiology , Neoplasms/therapy , Antigens, Neoplasm/chemistry , Carbonic Anhydrase IX , Carbonic Anhydrases/chemistry , Gene Expression , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Molecular Targeted Therapy , Neoplasms/enzymology , Structural Homology, Protein
16.
J Med Chem ; 57(22): 9673-86, 2014 Nov 26.
Article in English | MEDLINE | ID: mdl-25358036

ABSTRACT

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.


Subject(s)
Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/chemistry , Ethers/chemistry , Glaucoma/drug therapy , Animals , Carbon Dioxide , Crystallography, X-Ray/methods , Disease Models, Animal , Drug Design , Fluorescent Dyes/chemistry , Humans , Kinetics , Male , Models, Chemical , Molecular Structure , Neoplasms/metabolism , Rabbits , Structure-Activity Relationship , Water/chemistry
17.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 10): 1324-7, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25286933

ABSTRACT

The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO2. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5 Šresolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO2-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (∼40 µM) were measured using (18)O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO2 substrate-binding site.


Subject(s)
Carbonic Anhydrase II/chemistry , Carbonic Anhydrase Inhibitors/chemistry , Cyanates/chemistry , Carbon Dioxide/chemistry , Catalytic Domain , Crystallography, X-Ray , Humans , Models, Molecular , Protein Binding
18.
Subcell Biochem ; 75: 31-52, 2014.
Article in English | MEDLINE | ID: mdl-24146373

ABSTRACT

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, ß, γ, δ and ζ). The α-class is found primarily in vertebrates (and the only class of CA in mammals), ß 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.


Subject(s)
Carbon Dioxide/chemistry , Carbonic Anhydrases/chemistry , Carbonic Anhydrases/genetics , Catalytic Domain , Animals , Archaea/enzymology , Bicarbonates/chemistry , Carbon Dioxide/metabolism , Carbonic Anhydrases/classification , Carbonic Anhydrases/metabolism , Catalysis , Plants/enzymology , Protein Conformation , Protons , Structure-Activity Relationship
19.
Bioorg Med Chem ; 21(22): 7210-5, 2013 Nov 15.
Article in English | MEDLINE | ID: mdl-24090602

ABSTRACT

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.


Subject(s)
Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrases/chemistry , Sulfonamides/chemistry , Thiazines/chemistry , Thiophenes/chemistry , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/metabolism , Binding Sites , Carbonic Anhydrase II/chemistry , Carbonic Anhydrase II/genetics , Carbonic Anhydrase II/metabolism , Carbonic Anhydrase IX , Carbonic Anhydrase Inhibitors/metabolism , Carbonic Anhydrases/metabolism , Catalytic Domain , Crystallography, X-Ray , Humans , Kinetics , Molecular Docking Simulation , Mutagenesis, Site-Directed , Protein Binding , Structure-Activity Relationship , Sulfonamides/metabolism , Thermodynamics , Thiazines/metabolism , Thiophenes/metabolism
20.
Article in English | MEDLINE | ID: mdl-23908036

ABSTRACT

Pseudomonas aeruginosa is a Gram-negative bacterium that causes life-threatening infections in susceptible individuals and is resistant to most clinically available antimicrobials. Genomic and proteomic studies have identified three genes, pa0102, pa2053 and pa4676, in P. aeruginosa PAO1 encoding three functional ß-carbonic anhydrases (ß-CAs): psCA1, psCA2 and psCA3, respectively. These ß-CAs could serve as novel antimicrobial drug targets for this pathogen. X-ray crystallographic structural studies have been initiated to characterize the structure and function of these proteins. This communication describes the production of two crystal forms (A and B) of ß-CA psCA3. Form A diffracted to a resolution of 2.9 Å; it belonged to space group P212121, with unit-cell parameters a = 81.9, b = 84.9, c = 124.2 Å, and had a calculated Matthews coefficient of 2.23 ų Da⁻¹ assuming four molecules in the crystallographic asymmetric unit. Form B diffracted to a resolution of 3.0 Å; it belonged to space group P21212, with unit-cell parameters a = 69.9, b = 77.7, c = 88.5 Å, and had a calculated Matthews coefficient of 2.48 ų Da⁻¹ assuming two molecules in the crystallographic asymmetric unit. Preliminary molecular-replacement solutions have been determined with the PHENIX AutoMR wizard and refinement of both crystal forms is currently in progress.


Subject(s)
Bacterial Proteins/chemistry , Carbonic Anhydrases/chemistry , Pseudomonas aeruginosa/enzymology , Bacterial Proteins/biosynthesis , Carbonic Anhydrases/biosynthesis , Crystallography, X-Ray
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