Evaluation of the Impact of Esterases and Lipases from the Circulatory System against Substrates of Different Lipophilicity

Esterases and lipases can process amphiphilic esters used as drugs and prodrugs and impact their pharmacokinetics and biodistribution. These hydrolases can also process ester components of drug delivery systems (DDSs), thus triggering DDSs destabilization with premature cargo release. In this study we tested and optimized assays that allowed us to quantify and compare individual esterase contributions to the degradation of substrates of increased lipophilicity and to establish limitations in terms of substrates that can be processed by a specific esterase/lipase. We have studied the impact of carbonic anhydrase; phospholipases A1, A2, C and D; lipoprotein lipase; and standard lipase on the hydrolysis of 4-nitrophenyl acetate, 4-nitrophenyl palmitate, DGGR and POPC liposomes, drawing structure–property relationships. We found that the enzymatic activity of these proteins was highly dependent on the lipophilicity of the substrate used to assess them, as expected. The activity observed for classical esterases was diminished when lipophilicity of the substrate increased, while activity observed for lipases generally increased, following the interfacial activation model, and was highly dependent on the type of lipase and its structure. The assays developed allowed us to determine the most sensitive methods for quantifying enzymatic activity against substrates of particular types and lipophilicity

The Phospholipase A2 Superfamily: Structure, Isozymes, Catalysis, Physiologic and Pathologic Roles

The phospholipase A2 (PLA2) superfamily of phospholipase enzymes hydrolyzes the ester bond at the sn-2 position of the phospholipids, generating a free fatty acid and a lysophospholipid. The PLA2s are amphiphilic in nature and work only at the water/lipid interface, acting on phospholipid assemblies rather than on isolated single phospholipids. The superfamily of PLA2 comprises at least six big families of isoenzymes, based on their structure, location, substrate specificity and physiologic roles. We are reviewing the secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2), lipoprotein-associated PLA2 (LpPLA2), lysosomal PLA2 (LPLA2) and adipose-tissue-specific PLA2 (AdPLA2), focusing on the differences in their structure, mechanism of action, substrate specificity, interfacial kinetics and tissue distribution. The PLA2s play important roles both physiologically and pathologically, with their expression increasing significantly in diseases such as sepsis, inflammation, different cancers, glaucoma, obesity and Alzheimer’s disease, which are also detailed in this review

Carbonic Anhydrase Inhibitors: Synthesis of Membrane-Impermeant Low Molecular Weight Sulfonamides Possessing in Vivo Selectivity for the Membrane-Bound versus Cytosolic Isozymes1

Aromatic/heterocyclic sulfonamides act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA; EC 4.2.1.1), but the presently available compounds do not generally discriminate between the 14 isozymes isolated in higher vertebrates. Thus, clinically used drugs from this class of pharmacological agents show many undesired side effects due to unselective inhibition of all CA isozymes present in a tissue/organ. Here we propose a new approach for the selective in vivo inhibition of membrane-bound versus cytosolic CA isozymes with a new class of positively charged, membrane-impermeant sulfonamides. This approach is based on the attachment of trisubstituted-pyridinium-methylcarboxy moieties (obtained from 2,4,6-trisubstituted-pyrylium salts and glycine) to the molecules of classical aromatic/heterocyclic sulfonamides possessing free amino, imino, hydrazino, or hydroxyl groups in their molecules. Efficient in vitro inhibition (in the nanomolar range) was observed with some of the new derivatives against three investigated CA isozymes:  i.e., hCA I, hCA II (cytosolic forms), and bCA IV (membrane-bound isozyme) (h = human isozyme; b = bovine isozyme). Due to their salt-like character, the new type of inhibitors reported here, unlike the classical, clinically used compounds (such as acetazolamide, methazolamide, and ethoxzolamide), are unable to penetrate through biological membranes, as shown by ex vivo and in vivo perfusion experiments in rats. The level of bicarbonate excreted into the urine of the experimental animals perfused with solutions of the new and classical inhibitors undoubtedly proved that:  (i) when using the new type of positively charged sulfonamides, only the membrane-bound enzyme (CA IV) was inhibited, whereas the cytosolic isozymes (CA I and II) were not affected; (ii) in the experiments in which the classical compounds (acetazolamide, benzolamide, etc.) were used, unselective inhibition of all CA isozymes (I, II, and IV) has been evidenced