Marie Lopez

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.

Sulfonamide linked neoglycoconjugates--a new class of inhibitors for cancer-associated carbonic anhydrases.

The contribution of membrane-bound carbonic anhydrases (CAs) to hypoxic tumor growth and progression in cancer implicates cancer-associated CAs as a promising drug target for oncology. In this paper, we present a new class of sulfonamide-linked neoglycoconjugate that was designed to selectively target and inhibit the extracellular domains of the cancer-relevant CA isozymes. We describe the application of novel, yet straightforward, chemistry toward the synthesis of inhibitors that comprise both S-glycosyl sulfenamides and S-glycosyl sulfonamides. We also present the CA inhibition profile of our new neoglycoconjugates, more specifically a library of 30 compounds (3-32) that were designed to optimize both SAR (structure-activity relationship) and SPR (structure-property relationship) characteristics. We show that our approach produces neutral, water-soluble, and potent inhibitors (K(i)s in the low nanomolar range) that target cancer-associated CAs.

S-glycosyl primary sulfonamides--a new structural class for selective inhibition of cancer-associated carbonic anhydrases.

In this paper, we present a new class of carbonic anhydrase (CA) inhibitor that was designed to selectively target the extracellular domains of the cancer-relevant CA isozymes. The aromatic moiety of the classical zinc binding sulfonamide CA inhibitors is absent from these compounds and instead they incorporate a hydrophilic mono- or disaccharide fragment directly attached to the sulfonamide group to give S-glycosyl primary sulfonamides (1-10). The inhibition properties of these compounds at the physiologically abundant human CA isozymes I and II and cancer-associated IX and XII were determined, and all compounds had moderate potency with K(i)s in the micromolar range. We present the crystal structures of anomeric sulfonamides 4, 7, and 10 and the sugar sulfamate drug topiramate in complex with human recombinant CA II. From these structures, we have obtained valuable insights into ligand-protein interactions of these novel carbohydrate-based sulfonamides with CA.

Inhibition of carbonic anhydrase isozymes with benzene sulfonamides incorporating thio, sulfinyl and sulfonyl glycoside moieties

A series of benzene sulfonamides incorporating thio, sulfinyl or sulfonyl glycoside moieties were synthesized. These glycoconjugates were investigated for their ability to inhibit the enzymatic activity of four human carbonic anhydrases (hCA): isozymes I, II and tumour-associated isozymes IX and XII. The oxidation state of the sulfur in the carbohydrate tail moiety did not influence either enzyme inhibition potency or isozyme selectivity even though presenting opportunities for differing interactions with the target isozymes.

Synthesis of S-glycosyl primary sulfonamides.

The synthesis of S-glycosyl sulfonamides wherein the primary sulfonamide functional group (-SO(2)NH(2)) is directly attached to the anomeric position of a carbohydrate moiety is reported. Our general approach consists of first introducing a thioacetate group at the anomeric center of a per-O-acetylated sugar derivative. From this follows formation of a glycosyl sulfenamide (sugar-SNR(2)), oxidation of the sulfenamide to give a glycosyl N-protected sulfonamide (sugar-SO(2)NR(2)), and removal of the sulfonamide protecting (R) group to yield a primary sulfonamide at the anomeric center (sugar-SO(2)NH(2)). A variety of mono- and disaccharide derivatives were synthesized using this new methodology.

Promiscuity of carbonic anhydrase II: Unexpected ester hydrolysis of carbohydrate-based sulfamate inhibitors

Carbonic anhydrases (CAs) are enzymes whose endogenous reaction is the reversible hydration of CO(2) to give HCO(3)(-) and a proton. CA are also known to exhibit weak and promiscuous esterase activity toward activated esters. Here, we report a series of findings obtained with a set of CA inhibitors that showed quite unexpectedly that the compounds were both inhibitors of CO(2) hydration and substrates for the esterase activity of CA. The compounds comprised a monosaccharide core with the C-6 primary hydroxyl group derivatized as a sulfamate (for CA recognition). The remaining four sugar hydroxyl groups were acylated. Using protein X-ray crystallography, the crystal structures of human CA II in complex with four of the sulfamate inhibitors were obtained. As expected, the four structures displayed the canonical CA protein-sulfamate interactions. Unexpectedly, a free hydroxyl group was observed at the anomeric center (C-1) rather than the parent C-1 acyl group. In addition, this hydroxyl group is observed axial to the carbohydrate ring while in the parent structure it is equatorial. A mechanism is proposed that accounts for this inversion of stereochemistry. For three of the inhibitors, the acyl groups at C-2 or at C-2 and C-3 were also absent with hydroxyl groups observed in their place and retention of stereochemistry. With the use of electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), we observed directly the sequential loss of all four acyl groups from one of the carbohydrate-based sulfamates. For this compound, the inhibitor and substrate binding mode were further analyzed using free energy calculations. These calculations suggested that the parent compound binds almost exclusively as a substrate. To conclude, we have demonstrated that acylated carbohydrate-based sulfamates are simultaneously inhibitor and substrate of human CA II. Our results suggest that, initially, the substrate binding mode dominates, but following hydrolysis, the ligand can also bind as a pure inhibitor thereby competing with the substrate binding mode.

Design, Synthesis, and Biological Evaluation of Novel Carbohydrate-Based Sulfamates as Carbonic Anhydrase Inhibitors

Carbonic anhydrases (CAs) IX and XII are enzymes with newly validated potential for the development of personalized, first-in-class cancer chemotherapies. Here we present the design and synthesis of novel carbohydrate-based CA inhibitors, several of which were very efficient inhibitors (K(i)<10 nM) with good selectivity for cancer-associated CA isozymes over off-target CA isozymes. All inhibitors comprised a carbohydrate core with one hydroxyl group derivatized as a sulfamate. Five different carbohydrates were chosen to present a selection of molecular shapes with subtle stereochemical differences to the CA enzymes active site. Variable modifications of the remaining sugar hydroxyl groups were incorporated to provide an incremental coverage of chemical property parameters that are associated with biopharmaceutical performance. All sulfamate inhibitors displayed ligand efficiencies that are consistent with those reported for good drug lead candidates.

Synthesis of acylated glycoconjugates as templates to investigate in vitro biopharmaceutical properties

A series of novel glycopyranosyl azides were synthesised wherein the carbohydrate moiety was peracylated with four acetyl, propionyl, butanoyl, pentanoyl (valeryl) or 3-methylbutanoyl (isovaleryl) ester linked groups. A panel of glycoconjugates was synthesised from these glycopyranosyl azides using copper-catalysed azide-alkyne cycloaddition. The in vitro metabolic stability, plasma stability and plasma protein binding was then measured to establish the impact of the different acyl group when presented on a common scaffold. The acetyl, propionyl and butanoyl esters exhibited metabolism consistent with esterase processing, and various mono-, di- and tri-acylated hydrolysis products as well as the fully hydrolysed compound were detected. In contrast, the pentanoyl and 3-methylbutanoyl esters were stable.

Design and synthesis of thiourea compounds that inhibit transmembrane anchored carbonic anhydrases

A library of 32 novel glycoconjugate thiourea-bridged benzene sulfonamides have been synthesized from the reaction of glycosyl isothiocyanates with a panel of simple benzene sulfonamides comprising either a free amine or hydrazide. All compounds were investigated for their ability to inhibit the enzymatic activity of five human carbonic anhydrase (hCA) isozymes: hCA I, II and membrane-associated isozymes IX, XII and XIV. A physicochemical feature of the free sugar thioureido glycoconjugates was high water solubility (> 20 mg/mL), as well many of these compounds exhibited a desirable potency and CA isozyme selectivity profile. From this library several inhibitors displayed excellent potency-selectivity profiles for transmembrane anchored CAs over off-target CA I and II. These molecules provide potential dual-acting candidates for the development of inhibitors that target the extracellular CAs (IX, XII and XIV)-either directly as free sugars (membrane impermeable) or indirectly as acetylated prodrugs, becoming free sugars upon esterase hydrolysis.

Antimalarial activity of compounds comprising a primary benzene sulfonamide fragment

Despite the urgent need for effective antimalarial drugs with novel modes of action no new chemical class of antimalarial drug has been approved for use since 1996. To address this, we have used a rational approach to investigate compounds comprising the primary benzene sulfonamide fragment as a potential new antimalarial chemotype. We report the in vitro activity against Plasmodium falciparum drug sensitive (3D7) and resistant (Dd2) parasites for a panel of fourteen primary benzene sulfonamide compounds. Our findings provide a platform to support the further evaluation of primary benzene sulfonamides as a new antimalarial chemotype, including the identification of the target of these compounds in the parasite.