Fundamental statistics in psychology and education.

1. Introduction: Overview of CA as a Model
Carbonic anhydrase (CA, EC 4.2.1.1) is a protein that is especially well-suited to serve as a model in many types of studies in biophysics, bioanalysis, the physical-organic chemistry of inhibitor design, and medicinal chemistry. In vivo, this enzyme catalyzes the hydration of CO2 and the dehydration of bicarbonate (eq 1).


CO2+H2O⇌HCO3−+H+

(1)


The active site of α-CAs comprises a catalytic ZnII ion coordinated by three imidazole groups of histidines and by one hydroxide ion (or water molecule), all in a distorted tetrahedral geometry. This grouping is located at the base of a cone-shaped amphiphilic depression, one wall of which is dominated by hydrophobic residues and the other of which is dominated by hydrophilic residues.1 Unless otherwise stated, “CA” in this review refers to (i) various isozymes of α-CAs or (ii) the specific α-CAs human carbonic anhydrases I and II (HCA I and HCA II) and bovine carbonic anhydrase II (BCA II); “HCA” refers to HCA I and HCA II; and “CA II” refers to HCA II and BCA II.

CA is particularly attractive for biophysical studies of protein–ligand binding for many reasons. (i) CA is a monomeric, single-chain protein of intermediate molecular weight (~30 kDa), and it has no pendant sugar or phosphate groups and no disulfide bonds. (ii) It is inexpensive and widely available. (iii) It is relatively easy to handle and purify, due in large part to its excellent stability under standard laboratory conditions. (iv) Amino acid sequences are available for most of its known isozymes. (v) The structure of CA, and of its active site, has been defined in detail by X-ray diffraction, and the mechanism of its catalytic activity is well-understood. (vi) As an enzyme, CA behaves not only as a hydratase/anhydrase with a high turnover number but also as an esterase (a reaction that is easy to follow experimentally). (vii) The mechanism of inhibition of CA by ligands that bind to the ZnII ion is fairly simple and well-characterized; it is, therefore, easy to screen inhibitors and to examine designed inhibitors that test theories of protein–ligand interactions. (viii) It is possible to prepare and study the metal-free apoenzyme and the numerous variants of CA in which the ZnII ion is replaced by other divalent ions. (ix) Charge ladders of CA II—sets of derivatives in which acylation of lysine amino groups (−NH3+ → −NHAc) changes the net charge of the protein—allow the influence of charge on properties to be examined by capillary electrophoresis. Some disadvantages of using CA include the following: (i) the presence of the ZnII cofactor, which can complicate biophysical and physical-organic analyses; (ii) a structure that is more stable than a representative globular protein and, thus, slightly suspect as a model system for certain studies of stability; (iii) a function—interconversion of carbon dioxide and carbonate—that does not involve the types of enzyme/substrate interactions that are most interesting in design of drugs; (iv) a catalytic reaction that is, in a sense, too simple (determining the mechanism of a reaction is, in practice, usually made easier if the reactants and products have an intermediate level of complexity); and (v) the absence of a solution structure of CA (by NMR spectroscopy). The ample X-ray data, however, paint an excellent picture of the changes (which are generally small) in the structure of CA that occur on binding ligands or introducing mutations.

The most important class of inhibitors of CA, the aryl-sulfonamides, has several characteristics that also make it particularly suitable for physical-organic studies of inhibitor binding and in drug design: (i) arylsulfonamides are easily synthesized; (ii) they bind with high affinity to CA (1 μM to sub-nM); (iii) they share one common structural feature; and (iv) they share a common, narrowly defined geometry of binding that exposes a part of the ligand that can be easily modified synthetically. There are also many non-sulfonamide, organic inhibitors of CA, as well as anionic, inorganic inhibitors.

We divide this review into five parts, all with the goal of using CA as a model system for biophysical studies: (I) an overview of the enzymatic activity and medical relevance of CA; (II) the structure and structure–function relationships of CA and its engineered mutants; (III) the thermodynamics and kinetics of the binding of ligands to CA; (IV) the effect of electrostatics on the binding of ligands to and the denaturation of CA; and (V) what makes CA a good model for studying protein–ligand binding and protein stability.



1.1. Value of Models 
CA serves as a good model system for the study of enzymes. That is, it is a protein having some characteristics representative of enzymes as a class, but with other characteristics that make it especially easy to study. It is a moderately important target in current medicinal chemistry: its inhibition is important in the treatment of glaucoma, altitude sickness, and obesity; its overexpression has recently been implicated in tumor growth; and its inhibition in pathogenic organisms might lead to further interesting drugs.2,3 More than its medical relevance, its tractability and simplicity are what make CA a particularly attractive model enzyme.

The importance of models in science is often underestimated. Models represent more complex classes of related systems and contribute to the study of those classes by focusing research on particular, tractable problems. The development of useful, widely accepted models is a critical function of scientific research: many of the techniques (both experimental and analytical) and concepts of science are developed in terms of models; they are thoroughly engrained in our system of research and analysis.

Examples of models abound in successful areas of science: in biology, E. coli, S. cerevisiae, Drosophila mela-nogaster, C. elegans, Brachydanio rerio (zebrafish), and the mouse; in chemistry, the hydrogen atom, octanol as a hydrophobic medium, benzene as an aromatic molecule, the 2-norbornyl carbocation as a nonclassical ion, substituted cyclohexanes for the study of steric effects, p-substituted benzoic acids for the study of electronic effects, cyclodextrins for ligand–receptor interactions; in physics, a vibrating string as an oscillator and a particle in a box as a model for electrons in orbitals.

Science needs models for many reasons:


Focus: Models allow a community of researchers to study a common subject. Solving any significant problem in science requires a substantial effort, with contributions from many individuals and techniques. Models are often the systems chosen to make this productive, cooperative focus possible.


Research Overhead: Development of a system to the point where many details are scientifically tractable is the product of a range of contributions: for enzymes, these contributions are protocols for preparations, development of assays, determination of structures, preparation of mutants, definition of substrate specificity, study of rates, and development of mechanistic models. In a well-developed model system, the accumulation of this information makes it relatively easy to carry out research, since before new experiments begin, much of the background work–the fundamental research in a new system–has already been carried out.


Recruiting and Interdisciplinarity: The availability of good model systems makes it relatively easy for a neophyte to enter an area of research and to test ideas efficiently. This ease of entry recruits new research groups, who use, augment, and improve the model system. It is especially important to have model systems to encourage participation by researchers in other disciplines, for whom even the elementary technical procedures in a new field may appear daunting.


Comparability: A well-established model allows researchers in different laboratories to calibrate their experiments, by reproducing well-characterized experiments.


Community: The most important end result of a good model system is often the generation of a scientific community–that is, a group of researchers examining a common problem from different perspectives and pooling information relevant to common objectives.



One of the goals of this review is to summarize many experimental and theoretical studies of CA that have established it as a model protein. We hope that this summary will make it easier for others to use this protein to study fundamentals of two of the most important questions in current chemistry: (i) Why do a protein and ligand associate selectively? (ii) How can one design an inhibitor to bind to a protein selectively and tightly? We believe that the summary of studies of folding and stability of CA will be useful to biophysicists who study protein folding. In addition, we hope that the compilation of data relevant to CA in one review will ease the search for information for those who are beginning to work with this protein.

/pdf/carbonic-anhydrase-as-a-model-for-biophysical-and-physical-1nijnawjpq.pdf

Carbonic Anhydrase as a Model for Biophysical and Physical-Organic Studies of Proteins and Protein–Ligand Binding

Discovered 80 year ago, in 1933, carbonic anhydrases (CAs) were intensely investigated due to the biomedical applications of their inhibitors and because these enzymes (characterized by the presenc...

https://www.tandfonline.com/doi/pdf/10.3109/14756366.2013.761876

Carbonic anhydrases: from biomedical applications of the inhibitors and activators to biotechnological use for CO(2) capture.

Introduction: Glaucoma is one of the major causes of blindness, affecting together with age-related macular degeneration > 70 million people worldwide. One of the therapeutic options for its management is based on the inhibition of the metalloenyme carbonic anhydrase (CA, EC 4.2.1.1). CA inhibitors (CAIs) diminish intraocular pressure (IOP) by reducing the rate of bicarbonate formation and thus secretion of the aqueous humor. Areas covered: The main classes of clinically used antiglaucoma CAIs are the sulfonamides with systemic (acetazolamide, methazolamide, ethoxzolamide and dichlorophenamide) and topical (dorzolamide and brinzolamide) action. A patent literature review covering the period 2007 – 2013 is presented. Expert opinion: This review presents an overview of the patent literature in the CAI antiglaucoma drug design field during the past 6 years. Most of the patents deal with sulfonamide/sulfamide/sulfamate CAIs, sulfonamides incorporating NO-donating moieties, as well as hybrids incorporating sul...

Antiglaucoma carbonic anhydrase inhibitors: a patent review.

Introduction: Obesity is ranked as one of the top 10 global health problems and the major concern deriving from it is the exposure of the population to a vast array of chronic pathologies such as cardiovascular and musculoskeletal disorders, type 2 diabetes, cancer, such as colon, breast and endometrial cancer, together with psychological disorders derived from this condition. The discovery that the clinically used anticonvulsants topiramate (TPM) and zonisamide (ZNS) induced weight loss in obese, epileptic patients, afforded the validation of the mitochondrial carbonic anhydrases (CAs, EC 4.2.1.1) VA and VB as targets for the development of antiobesity drugs. Areas covered: This review deals with the scientific and patent literature regarding obesity or obesity-related pathologies, being particularly focused on the use of carbonic anhydrase inhibitors (CAI) such as TPM and ZNS which inhibit the de novo lipogenesis. Expert opinion: There is an urgent need of new drugs for the treatment of obesity. The ide...

Antiobesity carbonic anhydrase inhibitors: a literature and patent review.

The human pathogen Mycobacterium tuberculosis contains three β-carbonic anhydrases (CAs, EC 4.2.1.1) in its genome. Inhibition of some of these CAs was shown to modulate the growth of M. tuberculosis. 3D-QSAR Comparative molecular field analyses (CoMFA) were carried out on inhibitors of the enzyme Rv3588c (also denominated mtCA 2). A series of sulfonamides known to inhibit mtCA 2, including some diazenylbenzenesulfonamides, was considered in our study. The predictive ability of the model was assessed using a test set of seven compounds. The best model has demonstrated a good fit having predictive r2 value of 0.93 and cross-validated coefficient q2 value as 0.88 in tripos CoMFA region. Our results indicate that the steric and electrostatic factors play a significant role in mtCA 2 inhibition for the investigated compounds. We proposed nine new not yet synthesized mtCA 2 inhibitors, all of them probably with significantly improved anti-Rv3588c inhibitory activity.

https://www.tandfonline.com/doi/pdf/10.3109/14756366.2013.800059

3D-QSAR CoMFA studies on sulfonamide inhibitors of the Rv3588c β-carbonic anhydrase from Mycobacterium tuberculosis and design of not yet synthesized new molecules

Comparative quantitative structure–activity relationship studies on para-substituted aromatic sulphonamides carbonic anhydrase II (CAII) inhibitors are reported in this paper. The study is made utilizing (i) information indices along; (ii) distance-based and connectivity indices and (iii) combination of information, distance-based and connectivity type topological indices. The study has shown that distance-based and connectivity type indices are superior for modelling, monitoring and estimating CAII inhibition. The results are critically discussed using a variety of statistical parameters. Our results show that starting from the mono-parametric regression itself, our results are superior: Furthermore, our methodology allowed carrying out much higher-parametric regressions, yielding a nine-parametric model with R 2 as high as 0.8375. The eightparametric regression, gave R 2 = 0.8343. As there is not much difference, we have considered the eight-parametric regression the best.

http://lori.academicdirect.org/cites/200803.pdf

Comparative QSAR Study on Para-substituted Aromatic Sulphonamides as CAII Inhibitors: Information versus Topological (Distance-Based and Connectivity) Indices

Shalini Singh

Papers

3D-QSAR CoMFA studies on sulfonamide inhibitors of the Rv3588c β-carbonic anhydrase from Mycobacterium tuberculosis and design of not yet synthesized new molecules

Comparative QSAR Study on Para-substituted Aromatic Sulphonamides as CAII Inhibitors: Information versus Topological (Distance-Based and Connectivity) Indices

Novel estimation of lipophilic behaviour of polychlorinated biphenyls

QSAR study on bioconcentration factor (BCF) of polyhalogented biphenyls using the PI index.

Use of the PI index in predicting toxicity of nitrobenzene derivatives.