Development of Polyamine Lassos as Polyamine Transport Inhibitors.

Nine- and twelve-membered triaza-macrocycles were appended to one end of homospermidine to make polyamine lassos. These compounds were shown to be potent polyamine transport inhibitors (PTIs) using pancreatic ductal adenocarcinoma L3.6pl cells, which have high polyamine transport activity. The smaller triazacyclononane-based lasso significantly reduced the uptake of a fluorescent polyamine probe and inhibited spermidine uptake and reduced intracellular polyamine levels in difluoromethylornithine (DFMO)-treated L3.6pl cells. Both designs were shown to be effective inhibitors of 3H-spermidine uptake, with the smaller lasso outperforming the larger lasso. When the smaller lasso was challenged to inhibit each of the three radiolabeled native polyamines, it had similar K i values as those of the known PTIs, Trimer44NMe and AMXT1501. Because of these promising properties, these materials may have future anticancer applications in polyamine blocking therapy, an approach that couples a polyamine biosynthesis inhibitor (DFMO) with a PTI to lower intracellular polyamines and suppress cell growth.

HappyTools: A software for high-throughput HPLC data processing and quantitation.

High-performance liquid chromatography (HPLC) is widely used for absolute quantitation. The advent of new columns and HPLC technology has enabled higher sample throughput, and hence, larger scale studies that perform quantitation on different sample types (e.g. healthy controls vs. patients with rheumatoid arthritis) using HPLC are becoming feasible. However, there remains a lack of methods that can analyse the increased number of HPLC samples. To address this in part, the modular toolkit HappyTools has been developed for the high-throughput targeted quantitation of HPLC measurements. HappyTools enables the user to create an automated workflow that includes retention time (tr) calibration, data extraction and the calculation of several quality criteria for data curation. HappyTools has been tested on a biopharmaceutical standard and previously published clinical samples. The results show comparable accuracy between HappyTools, Waters Empower and ThermoFisher Chromeleon. However, HappyTools offered superior precision and throughput when compared with Waters Empower and ThermoFisher Chromeleon. HappyTools is released under the Apache 2.0 license, both the source code and a Windows binary can be freely downloaded from https://github.com/Tarskin/HappyTools.

Synthesis and transfection efficiencies of new lipophilic polyamines.

A homologous series of lipophilic polyamines was synthesized and evaluated for DNA delivery and transfection efficiency. The series contained 1,4-butanediamine, 1,8-octanediamine, 2-[2-(2-amino-ethoxy)-ethoxy]-ethylamine, homospermidine, and homospermine covalently attached via their N1 terminus to a 3,4-bis(oleyloxy)-benzyl motif. In addition, homospermidine and homospermine were also attached via amide linkers. The homospermidine derivatives (i.e., benzyl tether 25 and benzamide tether 27) showed a 3-fold and 4-fold respective enhancement in delivery of AlexaFluor-488-labeled DNA over the butanediamine analogue 22. Homospermine derivative 26 was shown to inhibit 14C-spermine uptake (IC50 approximately 10 microM), which implied that 26 is able to compete effectively for polyamine recognition sites on the cell surface. This study demonstrated that the number and position of the positive charges along the polyamine scaffold plays a key role in DNA delivery and in determining the transfection efficiency.

N1-substituent effects in the selective delivery of polyamine conjugates into cells containing active polyamine transporters.

Several N(1)-arylalkylpolyamines containing various aromatic ring systems were synthesized as their respective HCl salts. The N(1)-substituents evaluated ranged in size from N(1)-benzyl, N(1)-naphthalen-1-ylmethyl, N(1)-2-(naphthalen-1-yl)ethyl, N(1)-3-(naphthalen-1-yl)propyl, N(1)-anthracen-9-ylmethyl, N(1)-2-(anthracen-9-yl)ethyl, N(1)-3-(anthracen-9-yl)propyl, and pyren-1-ylmethyl. The polyamine architecture was also altered and ranged from diamine to triamine and tetraamine systems. Biological activities in L1210 (murine leukemia), Chinese hamster ovary (CHO), and CHO's polyamine transport-deficient mutant (CHO-MG) cell lines were investigated via IC(50) cytotoxicity determinations. K(i) values for spermidine uptake were also determined in L1210 cells. The size of the N(1)-arylalkyl substituent as well as the polyamine sequence used had direct bearing on the observed cytotoxicity profiles. N(1)-Tethers longer than ethylene showed dramatic loss of selectivity for the polyamine transporter (PAT) as shown in a CHO/CHO-MG cytotoxicity screen. In summary, there are clear limits to the size of N(1)-substituents, which can be accommodated by the polyamine transporter. A direct correlation was observed between polyamine-conjugate uptake and cytotoxicity. In this regard, a cytotoxicity model was proposed, which describes a hydrophobic pocket of set dimensions adjacent to the putative PAT polyamine-binding site.

Total synthesis of petrobactin and its homologues as potential growth stimuli for Marinobacter hydrocarbonoclasticus, an oil-degrading bacteria.

A modular synthesis was developed to access petrobactin, a catechol-containing siderophore isolated from Marinobacter hydrocarbonoclasticus. A range of petrobactin homologues with differing dihydroxybenzamide motifs and in one case an increased number of carbons in the polyamine backbone were also synthesized. As such, these systems represent new isomeric probes to study iron transport properties in M. hydrocarbonoclasticus. The synthesis of petrobactin and its homologues and the first biological study of how these agents influence the growth of Mycobacterhydrocarbonoclasticus are reported. New synthetic methods were developed to overcome issues (imide formation) encountered in earlier syntheses. Both the (1)H and (13)C NMR of petrobactin were consistent with the recently revised structure showing that petrobactin in fact contains a 3,4-dihydroxybenzene motif rather than a 2,3-dihydroxybenzene motif. The preliminary biological studies suggested that using the native petrobactin 1b for M. hydrocarbonoclasticus-specific growth stimulation may be a poor strategy for oil-spill cleanup.

Defining the molecular requirements for the selective delivery of polyamine conjugates into cells containing active polyamine transporters.

Several N(1)-substituted polyamines containing various spacer units between nitrogen centers were synthesized as their respective HCl salts. The N(1)-substituents included benzyl, naphthalen-1-ylmethyl, anthracen-9-ylmethyl, and pyren-1-ylmethyl. The polyamine spacer units ranged from generic (4,4-triamine, 4,3-triamine, and diaminooctane) spacers to more exotic [2-(ethoxy)ethanoxy-containing diamine, hydroxylated 4,3-triamine, and cyclohexylene-containing triamine] spacers. Two control compounds were also evaluated: N-(anthracen-9-ylmethyl)-butylamine and N-(anthracen-9-ylmethyl)-butanediamine. Biological activities in L1210 (murine leukemia), alpha-difluoromethylornithine (DFMO)-treated L1210, and Chinese hamster ovary (CHO) and its polyamine transport-deficient mutant (CHO-MG) cell lines were investigated via IC(50) cytotoxicity determinations. K(i) values for spermidine uptake were also determined in L1210 cells. Of the series studied, the N(1)-benzyl-4,4-triamine system 6 had significantly higher IC(50) values (lower cytotoxicity) in the L1210, CHO, and CHO-MG cell lines. A cellular debenzylation process was observed in L1210 cells with 6 and generated "free" homospermidine. The size of the N(1)-arylmethyl substituent had direct bearing on the observed cytotoxicity in CHO-MG cells. The N(1)-naphthalenylmethyl, N(1)-anthracenylmethyl, and N(1)-pyrenylmethyl 4,4-triamines had similar toxicity (IC(50)s: approximately 0.5 microM) in CHO cells, which have an active polyamine transporter (PAT). However, this series had IC(50) values of >100 microM, 66.7 microM, and 15.5 microM, respectively, in CHO-MG cells, which are PAT-deficient. The observed lower cytotoxicity in the PAT-deficient CHO-MG cell line supported the premise that the conjugates use PAT for cellular entry. In general, moderate affinities for the polyamine transporter were observed for the N-arylmethyl 4,4-triamine series with their L1210 K(i) values all near 3 microM. In summary, the 4,4-triamine motif was shown to facilitate entry of polyamine conjugates into cells containing active polyamine transporters.