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.

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

Molecular descriptors are widely employed to present molecular characteristics in cheminformatics. Various molecular-descriptor-calculation software programs have been developed. However, users of those programs must contend with several issues, including software bugs, insufficient update frequencies, and software licensing constraints. To address these issues, we propose Mordred, a developed descriptor-calculation software application that can calculate more than 1800 two- and three-dimensional descriptors. It is freely available via GitHub. Mordred can be easily installed and used in the command line interface, as a web application, or as a high-flexibility Python package on all major platforms (Windows, Linux, and macOS). Performance benchmark results show that Mordred is at least twice as fast as the well-known PaDEL-Descriptor and it can calculate descriptors for large molecules, which cannot be accomplished by other software. Owing to its good performance, convenience, number of descriptors, and a lax licensing constraint, Mordred is a promising choice of molecular descriptor calculation software that can be utilized for cheminformatics studies, such as those on quantitative structure–property relationships.

/pdf/mordred-a-molecular-descriptor-calculator-4jqsz8r4bi.pdf

Mordred: a molecular descriptor calculator

Health safety issues of synthetic food colorants.

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP- ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knock- out animals (PARS2/2 ) and corresponding wild-type animals (PARS1/1 ), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Exposure of embryonic fibroblasts from the PARS1/1 animals to per- oxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS 1/1 cells against the suppression of mitochondrial respiration in response to per- oxynitrite (50-100 mM). Similarly to PARS inhibition with INH2BP, the PARS 2/2 cells were protected against peroxyni- trite-induced injury. The protection against cellular injury by PARS2/2 phenotype or INH2BP waned when cells were chal- lenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS 2/2 phenotype reduced inducible nitric-oxide synthase (iNOS; EC 1.14.13.39) mRNA levels and inhibited production of NO in immunostimulated cells. INH2BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS2/2 cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxyni- trite formation, was found in the inf lamed joints. Oral treat- ment with INH2BP (0.5 gykg, daily), starting at the onset of arthritis (day 25), delayed the development of the clinical signs at days 26-35 and improved histological status in the knee and paw. Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protects against oxidant-induced cellular injury in vitro and exhibits anti-inf lammatory effects in vivo.

Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase (nitric oxideysuperoxideyinf lammationyinducible nitric-oxide synthaseyDNA single-strand break)

Unidad de Investigación de Diseño de Farmacos y Conectividad Molecular, Departamento de Quı́mica Fisica, Facultad de Farmacı́a, Universitat de València, 46100 Burjassot, València, Spain, Instituto de Tecnologia Quimica, CSIC-Universidad Politecnica de Valencia, Av. de los Naranjos s/n, 46022 València, Spain, and Dipartimento di Chimica, Università della Calabria, via P. Bucci 14/C, 87036 Rende (CS), Italy

Some new trends in chemical graph theory.

A novel, distance-cum-adjacency topological descriptor, termed as eccentric connectivity index, has been conceptualized, and its discriminating power has been investigated with regard to physical/biological properties of molecules. Correlation coefficients ranging from 95% to 99% were obtained using eccentric connectivity index in various datasets with regard to physical properties of diverse nature. These correlations were far superior to those correspondingly derived from the Wiener index. For structure−activity studies, a dataset, comprised of 94 substituted piperidinyl methyl ester and methylene methyl ester analogs as analgesic agents, was selected. Values of the eccentric connectivity index, the Wiener index, and Randic's molecular connectivity index were calculated, and active ranges were identified. Good correlations between topological descriptors and analgesic activity of these analogs were obtained. Eccentric connectivity index exhibited highest predictibility of the order of 86%. High discrimi...

Eccentric connectivity index : a novel highly discriminating topological descriptor for structure-property and structure-activity studies

Application of Graph Theory: Relationship of Eccentric Connectivity Index and Wiener's Index with Anti-inflammatory Activity

Eccentric Connectivity Index: A Novel Highly Discriminating Topological Descriptor for Structure-Property and Structure-Activity Studies

A simple, adjacency-cum-path length based, topological descriptor termed the connective eccentricity index has been conceptualized and its discriminating power investigated with regard to antihypertensive activity. A data set consisting of 81 derivatives of N-benzylimidazole was selected for the present investigation. These derivatives are potent, competitive, and nonpeptide angiotensin II receptor antagonists. The value of connective eccentricity index for each derivative was computed and active range identified. Subsequently, each derivative was assigned a biological activity that was compared with the reported antihypertensive activity. The results obtained using connective eccentricity index were better than the corresponding values obtained using Balaban’s mean square distance index. The accuracy of prediction was found to be about 80% in the active range using connective eccentricity index.

Anil Kumar Madan

Papers

Eccentric connectivity index : a novel highly discriminating topological descriptor for structure-property and structure-activity studies

Application of Graph Theory: Relationship of Eccentric Connectivity Index and Wiener's Index with Anti-inflammatory Activity

Eccentric Connectivity Index: A Novel Highly Discriminating Topological Descriptor for Structure-Property and Structure-Activity Studies

Connective eccentricity index: a novel topological descriptor for predicting biological activity.

Eccentric distance sum: A novel graph invariant for predicting biological and physical properties