Abstract
Serine proteases have proven to be promising pharmacological targets in contemporary drug discovery for cancer treatment. Since azaphenylalanine-based compounds manifest cytotoxic activity, we have selected serine protease inhibitors designed and synthesized in-house with large hydrophobic naphthalene moiety for screening. The cytotoxic potential of screened molecules was correlated to modifications of R1 residues. The most cytotoxic were compounds with greater basicity; amidinopiperidines, piperidines and benzamidines. Amidinopiperidine-based compounds exert cytotoxicity in low µM range, with IC50 18 µM and 22 µM for inhibitors 15 and 16 respectively. These compounds exhibited selective cytotoxicity towards the Burkitt’s lymphoma cells Ramos and Daudi, and proved nontoxic to PMBC, Jurkat and U937. They induce caspase-dependent apoptotic cell death, as demonstrated by the use of a pan-caspase inihibitor, zVADfmk, which was able to rescue Ramos cells from compound(s)-induced apoptosis. We confirm a disruption of the pro-survival pathway in Burkitt’s lymphoma through NFκB inhibition. The accumulation of phosphorylated precursor (p105) and inhibitory (IκB) molecules with no subsequent release of active NFκB implicated the involvement of proteasome. Indeed, we show that the amidinopiperidine-based compounds inhibit all three proteolytical activities of the human 20S proteasome, with the most prominent effect being on the trypsin-like activity. Consistently, treatment of Ramos cells with these compounds led to an increase in ubiquitinated proteins. The amidinopiperidine-based serine protease inhibitors presented are, as selective inducers of apoptosis in Burkitt’s lymphoma cells, promising leads for the development of novel chemotherapeutics.
Highlights
Apoptosis is a natural process essential for multicellular development and the maintenance of tissue homeostasis
In the search for novel inducers of apoptosis we have assayed selected serine protease inhibitors possessing N’acyl-2-naphthohydrazide or N’-acyl-naphthalene-2-sulfonohydrazide moiety, since we have previously shown that azaphenylalanine-based compounds show cytotoxic activity and concluded that a large hydrophobic naphthalene ring is crucial for the observed effect [24]
Our former investigations of apoptosis-inducing serine protease inhibitors revealed the cytotoxic effects of azaphenylalanine-based entities
Summary
Apoptosis is a natural process essential for multicellular development and the maintenance of tissue homeostasis. The development of drugs able to restore cell death may be an effective approach in the treatment of cancer [1,2]. Numerous proteins, including protein kinases, signalling adapters and proteases, have proven to be effective targets. Serine proteases form a large family of proteolytic enzymes involved in numerous biological processes and can be divided into three subgroups, depending on the specific substrate cleavage; the chymotrypsin-, trypsin- and elastase-like serine proteases. A limited number of serine proteases have been identified as actively participating in the process of cell death, among them granzymes A and B, HtrA2/Omi, apoptotic protein 24 (AP24) and tissue-type plasminogen activator (tPA) [8,9,10]. Studies with synthetic and endogenous serine protease inhibitors (serpins) have revealed that serine proteases may act as both the pro- and anti-apoptotic molecules [11,12]
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