Mayank Aggarwal

Dithiocarbamates strongly inhibit carbonic anhydrases and show antiglaucoma action in vivo

A series of dithiocarbamates were prepared by reaction of primary/secondary amines with carbon disulfide in the presence of bases. These compounds were tested for the inhibition of four human (h) isoforms of the zinc enzyme carbonic anhydrase, CA (EC 4.2.1.1), hCA I, II, IX, and XII, involved in pathologies such as glaucoma (CA II and XII) or cancer (CA IX). Several low nanomolar inhibitors targeting these CAs were detected. The X-ray crystal structure of the hCA II adduct with morpholine dithiocarbamate evidenced the inhibition mechanism of these compounds, which coordinate to the metal ion through a sulfur atom from the dithiocarbamate zinc-binding function. Some dithiocarbamates showed an effective intraocular pressure lowering activity in an animal model of glucoma.

Structural annotation of human carbonic anhydrases.

Carbonic anhydrases (CAs, EC 4.2.1.1) are a family of metalloenzymes that catalyze the reversible interconversion of CO2 and HCO3−. Of the 15 isoforms of human (h) α-CA, 12 are catalytic (hCAs I-IV, VA, VB, VI, VII, IX, XII-XIV). The remaining three acatalytic isoforms (hCAs VIII, X and XI) lack the active site Zn2+ and are referred to as CA-related proteins (CA-RPs); however, their function remains elusive. Overall these isoforms are very similar to each other in structure but they differ in their expression and distribution. The favourable properties of hCA II such as fast kinetics, easy expression and purification, high solubility and intermediate heat resistance have made it an attractive candidate for numerous industrial applications. This review highlights the structural similarity and stability comparison among hCAs.

Dithiocarbamates: a new class of carbonic anhydrase inhibitors. Crystallographic and kinetic investigations

The zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) is inhibited by several classes of zinc-binders (sulfonamides, sulfamates, and sulfamides) as well as by compounds which do not interact with the metal ion (phenols, polyamines and coumarins). Here we report a new class of potent CA inhibitors which bind the zinc ion: the dithiocarbamates (DTCs). They coordinate to the zinc ion from the enzyme active site in monodentate manner and establish many favorable interactions with amino acid residues nearby. Several low nanomolar CA I, II and IX inhibitors were detected.

Anticonvulsant/antiepileptic carbonic anhydrase inhibitors: a patent review.

Introduction: An epileptic seizure is a transient occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. The International League Against Epilepsy classifies seizures in two broad categories: partial (localized to one cerebral hemisphere) and generalized (localized to both cerebral hemispheres). One indirect pathway for the treatment of epilepsy includes the inhibition of carbonic anhydrase (CA), thereby increasing CO2 levels in the brain. Areas covered: Carbonic anhydrases (EC 4.2.1.1) are ubiquitous metalloenzymes that catalyze the reversible hydration/dehydration of CO2/HCO3 -, respectively. CA inhibitors (CAIs) such as acetazolamide, methazolamide, topiramate, zonisamide, and sulthiame can reduce seizures through perturbation of the CO2 equilibrium and/or the inhibition of ion channels. This review focuses on the mechanism of epilepsy, CA catalysis, and recent developments in the treatment of epilepsy using CAIs. Expert opinion: Based on the observed active-site binding interactions of CAIs in crystal structures and their respective inhibition constants, structure–activity relationships can be mapped. Various CAIs along with novel techniques to administer them have been patented in the last four years. However, epilepsy continues to be a path less traveled when it comes to CAIs. A major area of research must focus toward the design of isoform-specific inhibitors using analogs of existing CAIs.

Update on carbonic anhydrase inhibitors: a patent review (2008 – 2011)

Introduction: Carbonic anhydrases (CA) are a family of zinc metalloenzymes (EC 4.2.1.1) found in all organisms, catalyzing the reversible reaction of CO2 hydration to bicarbonate and a proton. CAs are involved in various physiological reactions including respiration, pH regulation, Na+ retention, calcification, tumorigenesis, electrolyte secretion, gluconeogenesis, ureagenesis, and lipogenesis. Hence CA inhibitors (CAIs) have long been studied as various classes of systemic anticonvulsants, anti-obesity, anti-pain, anti-tumor and topically acting anti-glaucoma agents, and agents for treating altitude sickness. In addition, CA isozyme IX (CA IX) has been shown to play a critical role in cancer proliferation, with CA IX overexpression in certain types of cancers leading to its use as a biomarker for cancer diagnosis and prognosis. Areas covered: Recently, the structures of all the catalytic α-CAs have been determined and with better understanding of gene-based RNA interference (RNAi) therapy, many patents on CA have been filed in the past few years. More new inhibitors are being tested in various labs around the world, in pursuit of designing high-affinity compounds that bind to specific isozymes of CA, thereby reducing side effects caused by off-target binding. This is a review on the patents filed related to CAs during 2008 – 2011. Expert opinion: Structural knowledge regarding CA isozymes offers great opportunity to explore and design inhibitors that will specifically bind to CA IX, avoid off-target binding with other CA isozymes and thus reduce side effects. Various in silico and kinetic studies to test and characterize novel and preexisting inhibitors for isozyme specificity are currently being conducted. However, there remains a need for more research to be carried out to develop better and more specific CAIs with lesser off-target inhibition.

Insights towards sulfonamide drug specificity in α-carbonic anhydrases

Carbonic anhydrases (CAs, EC 4.2.1.1) are a group of metalloenzymes that play important roles in carbon metabolism, pH regulation, CO2 fixation in plants, ion transport etc., and are found in all eukaryotic and many microbial organisms. This family of enzymes catalyzes the interconversion of CO2 and HCO3(-). There are at least 16 different CA isoforms in the alpha structural class (α-CAs) that have been isolated in higher vertebrates, with CA isoform II (CA II) being ubiquitously abundant in all human cell types. CA inhibition has been exploited clinically for decades for various classes of diuretics and anti-glaucoma treatment. The characterization of the overexpression of CA isoform IX (CA IX) in certain tumors has raised interest in CA IX as a diagnostic marker and drug target for aggressive cancers and therefore the development of CA IX specific inhibitors. An important goal in the field of CA is to identify, rationalize, and design potential compounds that will preferentially inhibit CA IX over all other isoforms of CA. The variations in the active sites between isoforms of CA are subtle and this causes non-specific CA inhibition which leads to various side effects. In the case of CA IX inhibition, CA II along with other isoforms of CA provide off-target binding sites which is undesirable for cancer treatment. The focus of this article is on CA IX inhibition and two different structural approaches to CA isoform specific drug designing: tail approach and fragment addition approach.

Lactate flux in astrocytes is enhanced by a non‐catalytic action of carbonic anhydrase II

Key points  •  Rapid exchange of metabolites like glucose and lactate between different cell types is crucial for energy supply to the brain. •  Carbonic anhydrase 2 (CAII) enhances lactate transport in mouse cerebellar and cerebral astrocytes. •  Enhancement of transport activity is independent of the enzymes catalytic function, but requires binding of CAII to the C‐terminal tail of the monocarboxylate transporter MCT1. •  CAII could enhance lactate flux by acting as a ‘proton collecting antenna’ for MCT1. •  By this mechanism CAII could enhance transfer of lactate between astrocytes and neurons and thus provide neurons with an increased supply of energy substrate.

Conformational variability of different sulfonamide inhibitors with thienyl-acetamido moieties attributes to differential binding in the active site of cytosolic human carbonic anhydrase isoforms

The X-ray crystal structures of the adducts of human carbonic anhydrase (hCA, EC 4.2.1.1) II complexed with two aromatic sulfonamides incorporating 2-thienylacetamido moieties are reported here. Although, the two inhibitors only differ by the presence of an additional 3-fluoro substituent on the 4-amino-benzenesulfonamide scaffold, their inhibition profiles against the cytosolic isoforms hCA I, II, III, VII and XIII are quite different. These differences were rationalized based on the obtained X-ray crystal structures, and their comparison with other sulfonamide CA inhibitors with clinical applications, such as acetazolamide, methazolamide and dichlorophenamide. The conformations of the 2-thienylacetamido tails in the hCA II adducts of the two sulfonamides were highly different, although the benzenesulfonamide parts were superimposable. Specific interactions between structurally different inhibitors and amino acid residues present only in some considered isoforms have thus been evidenced. These findings can explain the high affinity of the 2-thienylacetamido benzenesulfonamides for some pharmacologically relevant CAs (i.e., isoforms II and VII) being also useful to design high affinity, more selective sulfonamide inhibitors of various CAs.

Water Networks in Fast Proton Transfer during Catalysis by Human Carbonic Anhydrase II.

Variants of human carbonic anhydrase II (HCA II) with amino acid replacements at residues in contact with water molecules in the active-site cavity have provided insights into the proton transfer rates in this protein environment. X-ray crystallography and (18)O exchange measured by membrane inlet mass spectrometry have been used to investigate structural and catalytic properties of variants of HCA II containing replacements of Tyr7 with Phe (Y7F) and Asn67 with Gln (N67Q). The rate constants for transfer of a proton from His64 to the zinc-bound hydroxide during catalysis were 4 and 9 μs(-1) for Y7F and Y7F/N67Q, respectively, compared with a value of 0.8 μs(-1) for wild-type HCA II. These higher values observed for Y7F and Y7F/N67Q HCA II could not be explained by differences in the values of the pK(a) of the proton donor (His64) and acceptor (zinc-bound hydroxide) or by the orientation of the side chain of the proton shuttle residue His64. They appeared to be associated with a reduced level of branching in the networks of hydrogen-bonded water molecules between proton shuttle residue His64 and the zinc-bound solvent molecule as observed in crystal structures at 1.5-1.6 Å resolution. Moreover, Y7F/N67Q HCA II is unique among the variants studied in having a direct, hydrogen-bonded chain of water molecules between the zinc-bound solvent and N(ε) of His64. This study provides the clearest example to date of the relevance of ordered water structure to rate constants for proton transfer in catalysis by carbonic anhydrase.

Kinetic and structural characterization of thermostabilized mutants of human carbonic anhydrase II

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.