Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) plays a pivotal role in thiopurine treatment outcomes. However, little has been known about its intracellular regulation. Here, we describe the effect of fluctuations in physiological levels of S-adenosyl-l-methionine (SAM) and related metabolites on TPMT activity levels in cell lines and erythrocytes from healthy donors. We determined higher TPMT activity in wild-type TPMT*1/*1 individuals with high SAM concentrations (n=96) compared to the low SAM level group (n=19; P(0.001). These findings confirm the results of our in vitro studies, which demonstrated that the restriction of l-methionine (Met) in cell growth media reversibly decreased TPMT activity and protein levels. Selective inhibition of distinct components of Met metabolism was used to demonstrate that SAM is implicitly responsible for direct post-translational TPMT stabilization. The greatest effect of SAM-mediated TPMT stabilization was observed in the case of wild-type TPMT*1 and variant *3C allozymes. In addition to TPMT genotyping, SAM may serve as an important biochemical marker in individualization of thiopurine therapy.
COBISS.SI-ID: 3210097
There is a pressing need for the development of novel adjuvants for human use.The minimal bioactive structure of bacterial peptidoglycan (PGN), muramyl dipeptide (MDP) and its derivative murabutide (MB), have long been known for their adjuvant activities. For this reason, a series of novel desmuramyl dipeptides have been designed and synthesized as part of our search for therapeutically useful MDP analogues. Since nucleotide oligomerization domain 2 (Nod2) is a putative receptor for MDP, we used engineered HEK293 cells overexpressing Nod2 to screen and validate our compounds for their Nod2-agonist activity. Their immunomodulatory properties were subsequently assessed in vitro, by evaluating their effect on proinflammatory cytokine production of phorbol 12-myristate 13-acetate (PMA)/ionomycin-stimulated human peripheral blood mononuclear cells (PBMC). Herein, we present novel desmuramyl dipeptides, the most active of them possessing immunoenhancing properties as a result of their potent Nod2-agonistic effect.
COBISS.SI-ID: 3274353
Serine proteases have proven to be promising pharmacological targets in contemporary drug discovery for cancer treatment. Since azaphenylalanine-basedcompounds 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 mM range, with IC50 18 mM and 22 mM 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 thepro-survival pathway in Burkittʼs lymphoma through NFkB inhibition. The accumulation of phosphorylated precursor (p105) and inhibitory (IkB) molecules with no subsequent release of active NFkB 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.
COBISS.SI-ID: 3286129