Synthesis of tritium-labeled Lu-PSMA-617: Alternative tool for biological evaluation of radiometal-based pharmaceuticals.

This project focuses on the generation and evaluation of functional alternatives to radiometal-based pharmaceuticals supporting basic research and the in vitro developmental phase. Employing robust tritium chemistry and non-radioactive metal surrogates in two synthetic and labeling strategies resulted in ([ring-3H]Nal)PSMA-617 and ([α,ß-3H]Nal)PSMA-617. In particular, ([α,ß-3H]Nal)Lu-PSMA-617 exhibited high radiolytic as well as metal-complex stability and was compared to the clinically-established radiopharmaceutical [177Lu]Lu-PSMA-617. The cell-based assays confirmed the applicability of ([α,ß-3H]Nal)Lu-PSMA-617 as a substitute of [177Lu]Lu-PSMA-617 in pre-clinical biological settings.

Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1).

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13α-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13α/ß-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1-overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13α/ß-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13α/ß-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13α-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13α-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [3H]2-bromo-13α-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13α-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1.

Lessons on the Sigma-1 Receptor in TNBS-Induced Rat Colitis: Modulation of the UCHL-1, IL-6 Pathway.

Inflammatory Bowel Disease (IBD) is an autoimmune ailment of the gastrointestinal (GI) tract, which is characterized by enhanced activation of proinflammatory cytokines. It is suggested that the sigma-1 receptor (σ1R) confers anti-inflammatory effects. As the exact pathogenesis of IBD is still unknown and treatment options are limited, we aimed to investigate the effects of σ1R in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis. To this end, male Wistar-Harlan rats were used to model colitic inflammation through the administration of TNBS. To investigate the effects of σ1R, Fluvoxamine (FLV, σ1R agonist) and BD1063 (σ1R antagonist) were applied via intracolonic administration to the animals once a day for three days. Our radioligand binding studies indicated the existence of σ1Rs as [3H](+)-pentazocine binding sites, and FLV treatment increased the reduced σ1R maximum binding capacity in TNBS-induced colitis. Furthermore, FLV significantly attenuated the colonic damage, the effect of which was abolished by the administration of BD1063. Additionally, FLV potentially increased the expression of ubiquitin C-terminal hydrolase ligase-1 (UCHL-1) and the levels of endothelial nitric oxide synthase (eNOS), and decreased the levels of interleukin-6 (IL-6) and inducible NOS (iNOS) expression. In summary, our study offers evidence for the anti-inflammatory potential of FLV and σ1R in experimental colitis, and our results present a promising approach to the development of new σ1R-targeted treatment options against IBD.

Steroidal ferrocenes as potential enzyme inhibitors of the estrogen biosynthesis.

The potential inhibitory effect of diverse triazolyl-ferrocene steroids on key enzymes of the estrogen biosynthesis was investigated. Test compounds were synthesized via copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes using our efficient methodology published previously. Inhibition of human aromatase, steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) activities was investigated with in vitro radiosubstrate incubations. Some of the test compounds were found to be potent inhibitors of the STS. A compound bearing ferrocenyl side chain on the C-2 displayed a reversible inhibition, whereas C-16 and C-17 derivatives displayed competitive irreversible binding mechanism toward the enzyme. 17α-Triazolyl-ferrocene derivatives of 17ß-estradiol exerted outstanding inhibitory effect and experiments demonstrated a key role of the ferrocenyl moiety in the enhanced binding affinity. Submicromolar IC50 and Ki parameters enroll these compounds to the group of the most effective STS inhibitors published so far. STS inhibitory potential of the steroidal ferrocenes may lead to the development of novel compounds able to suppress in situ biosynthesis of 17ß-estradiol in target tissues.

Synthesis and structure-activity relationships of 2- and/or 4-halogenated 13ß- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis.

Ring A halogenated 13α-, 13ß-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17ß-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure-activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17ß-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.

Synthesis, receptor binding studies, optical spectroscopic and in silico structural characterization of morphiceptin analogs with cis-4-amino-L-proline residues.

Three novel morphiceptin analogs, in which Pro in position 2 and/or 4 was replaced by cis-4-aminoproline connected with the preceding amino acid through the primary amino group, were synthesized. The opioid receptor affinities, functional assay results, enzymatic degradation studies and experimental and in silico structural analysis of such analogs are presented. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins. Because GPI-anchored proteins can be reinserted into the cell membrane with the retention of the biological function, they are appropriate candidates for preparing models via reduction of the structural complexity. A synthetic headgroup was added to the 3ß-hydroxyl group of cholesterol, an essential lipid component of rafts, and the resulting cholesterol derivative was used as a simplified GPI mimetic. In order to quantitate the membrane integrated GPI mimetic after the exogenous addition to live cells, a tritium labelled cholesterol anchor was prepared. The radioactive label was introduced into the headgroup, and the radiolabelled GPI mimetic anchor was obtained with a specific activity of 1.37 TBq/mmol. The headgroup labelled cholesterol derivative was applied to demonstrate the sensitive detection of the cell membrane association of the anchor under in vivo conditions.

Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP1-7 in the rat spinal cord.

Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors.

In vitro quantitative study of the degradation of endomorphins.

The catabolism of the endomorphins was investigated in detail. The endomorphins were degraded relatively slowly in the rat brain homogenate (t1/2(endomorphin-1)=4.94 min; t1/2(endomorphin-2)=3.81 min). The inhibition of metalloproteases and aminopeptidases stabilised the endomorphins to the greatest extent. The digestion of endomorphins tritiated specifically on Tyr(1), Pro(2) or Phe(3) established also that only the aminopeptidase pathways were essential for inactivation of the endomorphins, and that the tetrapeptides were degraded by cleavage of the Pro(2)-Trp(3) or Pro(2)-Phe(3) bond. The end-products of the catabolism were amino acids; the fragments Tyr-Pro-OH and Pro-Trp-Phe-NH2 were present as intermediates. Metabolites produced by brain carboxypeptidases were not detected.