A mechanistic and kinetic study of the beta-lactone hydrolysis of Salinosporamide A (NPI-0052), a novel proteasome inhibitor

The aim of the present study was to investigate the mechanism of aqueous degradation of Salinosporamide A (NPI-0052; 1), a potent proteasome inhibitor that is currently in Phase I clinical trials for the treatment of cancer and is characterized by a unique beta-lactone-gamma-lactam bicyclic ring structure. The degradation of 1 was monitored by HPLC and by both low- and high-resolution mass spectral analyses. Apparent first-order rate constants for the degradation at 25 degrees C were determined in aqueous buffer solutions (ionic strength 0.15 M adjusted with NaCl) at various pH values in the range of 1 to 9. Degradation kinetics in water and in deuterium oxide were compared as a mechanistic probe. The studies were performed at pH (pD) 4.5 at 25 degrees C. To further confirm the reaction mechanism, the degradation was also performed in O-18-enriched water and the degradation products subjected to HPLC separation prior to mass spectral analysis. Solubility and stability in (SBE)(7m)-beta-cyclodextrin (Captisol) solutions were also determined. The hydrolytic degradation of 1, followed by both HPLC and LC/MS, showed that the drug in aqueous solutions gives a species with a molecular ion consistent with the beta-lactone hydrolysis product (NPI-2054; 2). This initial degradant further rearranges to a cyclic ether (NPI-2055; 3) via an intramolecular nucleophilic displacement reaction. The kinetic results showed that the degradation of 1 was moderately buffer catalyzed (general base) and the rate constants were pH independent in the range of 1-5 and base dependent above pH 6.5. No acid catalysis was observed. The kinetic deuterium solvent isotope effect (KSIE) was 3.1 (k(H)/k(D)) and a linear proton inventory plot showed that the rate-determining step involved only a single proton transfer. This suggested that a neighboring hydroxyl group (as opposed to a second water molecule) facilitated water attack at pD 4.5. Mass spectral analysis from the O-18-labeling studies proved that the mechanism involves acyl-oxygen bond cleavage and not a carbonium ion mechanism. 1 is unstable in water (t(90%) <= 33 min at pH <5) and degrades via beta-lactone hydrolysis involving a normal ester hydrolysis mechanism (addition-elimination) resulting in acyl-oxygen bond cleavage. Captisol solubilized and stabilized 1 in aqueous solutions. (c) 2007 Wiley-Liss, Inc

Ca2+ /S100 regulation of giant protein kinases

Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated(1). One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM)(2). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown(3). The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere(4). Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure(2,5-9)