Sally-Ann Poulsen

Non-Zinc Mediated Inhibition of Carbonic Anhydrases: Coumarins Are a New Class of Suicide Inhibitors

The X-ray crystal structure of the adduct between the zinc metalloenzyme carbonic anhydrase II (CA, EC 4.2.1.1) with the recently discovered natural product coumarin derivative 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one showed the coumarin hydrolysis product, a cis-2-hydroxy-cinnamic acid derivative, and not the parent coumarin, bound within the enzyme active site. The bound inhibitor exhibits an extended, two-arm conformation that effectively plugs the entrance to the enzyme active site with no interactions with the catalytically crucial zinc ion. The inhibitor is sandwiched between Phe131, with which it makes an edge-to-face stacking, and Asn67/Glu238sym, with which it makes several polar and hydrogen bonding interactions. This unusual binding mode, with no interactions between the inhibitor molecule and the active site metal ion is previously unobserved for this enzyme class and presents a new opportunity for future drug design campaigns to target a mode of inhibition that differs substantially from classical inhibitors such as the clinically used sulfonamides and sulfamates. Several structurally simple coumarin scaffolds were also shown to inhibit all 13 catalytically active mammalian CA isoforms, with inhibition constants ranging from nanomolar to millimolar. The inhibition is time dependent, with maximum inhibition being observed after 6 h.

Adenosine receptors: new opportunities for future drugs.

This review summarises current knowledge on adenosine receptors, an important G protein-coupled receptor. The four known adenosine receptor subtypes A1, A2A, A2B, and A3 are discussed with special reference to the opportunities for drug development.

Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum--the η-carbonic anhydrases.

The genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) thought to belong to the α-class, PfCA. Here we demonstrate the kinetic properties of PfCA for the CO2 hydration reaction, as well as an inhibition study of this enzyme with inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, and phenylboronic/arsonic acids, detecting several low micromolar inhibitors. A closer examination of the sequence of this and the CAs from other Plasmodium spp., as well as a phylogenetic analysis, revealed that these protozoa encode for a yet undisclosed, new genetic family of CAs termed the η-CA class. The main features of the η-CAs are described in this report.

Molecular evolution: dynamic combinatorial libraries, autocatalytic networks and the quest for molecular function

The principles of Darwinian evolution have been explored in molecular systems such as autocatalytic networks and dynamic combinatorial libraries. Molecular evolution in such systems manifests itself as ligand or receptor amplification by selection. Research efforts exploring these concepts may provide a mechanism for the identification of novel catalysts, molecular receptors and bioactive molecules.

Therapeutic applications of glycosidic carbonic anhydrase inhibitors

The zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1) are very efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate and hence play an important physiological role. In humans, 16 different isozymes have been described, some of them being involved in various pathological disorders. Several of these isozymes are considered as drug targets, and the design of selective inhibitors is a long‐standing goal that has captured the attention of researchers for 40 years and has lead to clinical applications against different pathologies such as glaucoma, epilepsy, and cancer. Among the different strategies developed for designing selective CA inhibitors (CAIs), the “sugar approach” has recently emerged as a new attractive and versatile tool. Incorporation of glycosyl moieties in different aromatic/heterocyclic sulfonamide/sulfamides/sulfamates scaffolds has led to the development of numerous and very effective inhibitors of potential clinical value. The clinical use of a highly active carbohydrate‐based CA inhibitor, i.e., topiramate, constitutes an interesting demonstration of the validity of this approach. Other carbohydrate‐based compounds also demonstrate promising potential for the treatment of ophthalmologic diseases. This review will focus on the development of this emerging sugar‐based approach for the development of CAIs.

Targeting Hypoxic Tumor Cell Viability with Carbohydrate-Based Carbonic Anhydrase IX and XII Inhibitors

Carbonic anhydrase (CA) enzymes, specifically membrane-bound isozymes CA IX and CA XII, underpin a pH-regulating system that enables hypoxic tumor cell survival and proliferation. CA IX and XII are implicated as potential targets for the development of new hypoxic cancer therapies. To date, only a few small molecules have been characterized in CA-relevant cell and animal model systems. In this paper, we describe the development of a new class of carbohydrate-based small molecule CA inhibitors, many of which inhibit CA IX and XII within a narrow range of low nanomolar K(i) values (5.3-11.2 nM). We evaluate for the first time carbohydrate-based CA inhibitors in cell-based models that emulate the protective role of CA IX in an acidic tumor microenvironment. Our findings identified two inhibitors (compounds 5 and 17) that block CA IX-induced survival and have potential for development as in vivo cancer cell selective inhibitors.

Natural Product-Based Phenols as Novel Probes for Mycobacterial and Fungal Carbonic Anhydrases

In order to discover novel probes that may help in the investigation and control of infectious diseases through a new mechanism of action, we have evaluated a library of phenol-based natural products (NPs) for enzyme inhibition against four recently characterized pathogen β-family carbonic anhydrases (CAs). These include CAs from Mycobacterium tuberculosis, Candida albicans, and Cryptococcus neoformans as well as α-family human CA I and CA II for comparison. Many of the NPs selectively inhibited the mycobacterial and fungal β-CAs, with the two best performing compounds displaying submicromolar inhibition with a preference for fungal over human CA inhibition of more than 2 orders of magnitude. These compounds provide the first example of non-sulfonamide inhibitors that display β over α CA enzyme selectivity. Structural characterization of the library compounds in complex with human CA II revealed a novel binding mode whereby a methyl ester interacts via a water molecule with the active site zinc.

Carbonic anhydrase inhibitors. Identification of selective inhibitors of the human mitochondrial isozymes VA and VB over the cytosolic isozymes I and II from a natural product-based phenolic library

We have investigated the enzyme inhibition characteristics of a natural product (NP)-based phenolic library against a panel of human carbonic anhydrases (hCAs, EC 4.2.1.1) which included hCAs I and II (cytosolic) and hCA VA/VB (mitochondrial isoforms). Most of these compounds were weak, micromolar inhibitors of the two cytosolic hCAs (K(I)s >10 microM) but showed good hCA VA/VB inhibitory activity with inhibition constants in the range of 70-125 nM. The selectivity ratios for inhibiting the mitochondrial over the cytosolic isoforms for these phenol derivatives were in the range of 120-3800, making them the most isoform-selective compounds for inhibiting hCA VA/VB known to date. The CA VA/VB enzymes are involved in biosynthetic processes such as gluconeogenesis, lipogenesis and ureagenesis, and no pharmacological inhibitors with good selectivity are currently available. Thus the NP inhibitors identified during these studies are excellent leads for obtaining even more effective compounds that selectively target mitochondrial hCAs, and also have the potential to be used as tools for understanding the physiological processes that are regulated by the two mitochondrial CA isoforms.

Direct Screening of a Dynamic Combinatorial Library Using Mass Spectrometry

A dynamic combinatorial library (DCL) screening approach is described that permits direct identification of the effective (from ineffective) combination of building blocks in the equilibrating DCL. The approach uses Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) together with sustained off-resonance irradiation collision activated dissociation (SORI-CAD) to detect noncovalent protein-DCL ligand complexes under native conditions. It was shown that in a single, rapid experiment one could concurrently identify all the ligands of interest from the DCL against a background of inactive DCL ligands while still in the presence of the target protein. This result has demonstrated that mass spectrometry may provide a fast preliminary screening approach to identify DCL candidates for later verification with more traditional but time-consuming analysis. The MS/MS enables DCL mixtures to be effectively deconvoluted without the need for either chromatography, synthesis of DCL sub-libraries, conversion of the DCL to a static library, or disruption of the protein-ligand complexes before analysis—all typically necessary for the current screening method for DCLs.

Natural product coumarins that inhibit human carbonic anhydrases

Natural products (NPs) have proven to be an invaluable source of new chemotherapies yet very few have been explored to source small molecule carbonic anhydrase (CA) inhibitors. CA enzymes underpin physiological pH and are critical to the progression of several diseases including cancer. The present study is the first to more widely investigate NP coumarins for CA inhibition following the recent discovery of a NP coumarin CA inhibitor. We assembled a NP library comprising 24 plant coumarins (compounds 4-27) and three ascidian coumarins (compounds 28-30) that together provide a diverse collection of structures containing the coumarin pharmacophore. This library was then evaluated for inhibition of six human CA isozymes (CAs I, II, VII, IX, XII and XIII) and a broad range of inhibition and isozyme selectivity profiles were evident. Our findings provide a platform to support further evaluation of NPs for the discovery of new chemotypes that inhibit disease relevant CA enzymes.