Irreversible inhibitors of serine proteases based on the \03B2-lactam scaffold as potential drug candidates

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), 2009, Universidade de Lisboa, Faculdade de FarmáciaDisponível no documentoCentro de Estudos de Ciências Farmacêuticas (CECF)/iMed.UL (Institute for Medicines and Pharmaceutical Sciences), Faculdade de Farmácia da Universidade de Lisboa. Fundação para a Ciência e a Tecnologia (SFRH/BD/17534/2004 e projecto PTDC/QUI/64056/2006

The efficiency of C-4 substituents in activating the -lactam scaffold towards serine proteases and hydroxide ion

The presence of a leaving group at C-4 of monobactams is usually considered to be a requirement for mechanism-based inhibition of human leukocyte elastase by these acylating agents. We report that second-order rate constants for the alkaline hydrolysis and elastase inactivation by N-carbamoyl monobactams are independent of the pKa of the leaving group at C-4. Indeed, the effect exerted by these substituents is purely inductive: electron-withdrawing substituents at C-4 of N-carbamoyl-3,3-diethylmonobactams increase the rate of alkaline hydrolysis and elastase inactivation, with Hammett rho-I values of 3.4 and 2.5, respectively, which indicate the development of a negative charge in the transition-states. The difference in magnitude between these rho-I values is consistent with an earlier transition-state for the enzymatic reaction when compared with that for the chemical process. These results suggest that rate limiting step in elastase inactivation is the formation of the tetrahedral intermediate, and that beta-lactam ring-opening is not concerted with the departure of a leaving group from C-4. Monobactam sulfones emerged as potent elastase inhibitors even when the ethyl groups at C-3, required for interaction with the primary recognition site, are absent. For one such compound, a 1:1 enzyme-inhibitor complex involving porcine pancreatic elastase has been examined by X-ray crystallography and shown to result from serine acylation and sulfinate departure from the β-lactam C-4

Irreversible inhibitors of serine proteases based on the \03B2-lactam scaffold as potential drug candidates

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), 2009, Universidade de Lisboa, Faculdade de FarmáciaDisponível no documentoCentro de Estudos de Ciências Farmacêuticas (CECF)/iMed.UL (Institute for Medicines and Pharmaceutical Sciences), Faculdade de Farmácia da Universidade de Lisboa. Fundação para a Ciência e a Tecnologia (SFRH/BD/17534/2004 e projecto PTDC/QUI/64056/2006

Crystallization and Preliminary Diffraction Studies of Porcine Pancreatic Elastase in Complex with a Novel Inhibitor

Porcine pancreatic elastase (PPE) was crystallized in complex with a novel inhibitor at pH 5 and X-ray diffraction data were collected at a synchrotron source to 1.66 Å. Crystals belong to the orthorhombic space group P212121, with unit cell parameters a = 50.25 Å, b = 57.94 Å and c = 74.69 Å. PPE is often used as model for drug target, due to its structural homology with the important therapeutic target human leukocyte elastase (HLE). Elastase is a serine protease that belongs to the chymotrypsin family, which has the ability to degrade elastin, an important component in connective tissues. Excessive elastin proteolysis leads to a number of pathological diseases

4-Oxo-β-lactams (Azetidine-2,4-diones) Are Potent and Selective Inhibitors of Human Leukocyte Elastase

Human leukocyte elastase (HLE) is a serine protease stored in and secreted from neutrophils that plays a determinant role in the pathogenesis of several lung diseases. 4-Oxo-β-lactams, previously reported as acylating agents of porcine pancreatic elastase, were found to be selective and potent inhibitors of HLE. Structure−activity relationship analysis showed that inhibitory activity is very sensitive to the nature of C-3 substituents, with small alkyl substituents such as a gem-diethyl group improving the inhibitory potency when compared to gem-methyl benzyl or ethyl benzyl counterparts. 4-Oxo-β-lactams containing a heteroarylthiomethyl group on the para position of an N1-aryl moiety afforded highly potent and selective inhibition of HLE, even at a very low inhibitor to enzyme ratio, as shown by the kon value of 3.24 × 106 M−1 s−1 for 6f. The corresponding ortho isomers were 40- to 90-fold less potent

Azetidine-2,4-diones (4-Oxo-β-lactams) as Scaffolds for Designing Elastase Inhibitors

A new class of inhibitors 4-oxo-β-lactams (azetidine-2,4-diones), containing the required structural elements for molecular recognition, inhibit porcine pancreatic elastase (PPE) but show a dramatically lower reactivity toward hydroxide compared with the analogous inhibitors 3-oxo-β-sultams. Inhibition is the result of acylation of the active site serine and electron-withdrawing substituents at the N-(4-aryl) position in 3,3-diethyl-N-aryl derivatives increasing the rate of enzyme acylation and generating a Hammett ρ-value of 0.65. Compared with a ρ-value of 0.96 for the rates of alkaline hydrolysis of the same series, this is indicative of an earlier transition state for the enzyme-catalyzed reaction. Docking studies indicate favorable noncovalent interactions of the inhibitor with the enzyme. Compound 2i, the most potent inhibitor against PPE, emerged as a very potent HLE inhibitor, with a second-order rate for enzyme inactivation of 5 × 105 M−1 s−1