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Supplemental material, sj-pdf-1-jbx-10.1177_24725552211030288 for ASCT1 and ASCT2: Brother and Sister? by Mariafrancesca Scalise, Lara Console, Jessica Cosco, Lorena Pochini, Michele Galluccio and Cesare Indiveri in SLAS Discovery

The large neutral amino acid transporter 1 (LAT1) is a promising anticancer target that is required for the cellular uptake of essential amino acids that serve as building blocks for cancer growth and proliferation. Here, we report a structure-based approach to identify chemically diverse and potent inhibitors of LAT1. First, a homology model of LAT1 that is based on the atomic structures of the prokaryotic homologs was constructed. Molecular docking of nitrogen mustards (NMs) with a wide range of affinity allowed for deriving a common binding mode that could explain the structure&minus, activity relationship pattern in NMs. Subsequently, validated binding hypotheses were subjected to molecular dynamics simulation, which allowed for extracting a set of dynamic pharmacophores. Finally, a library of ~1.1 million molecules was virtually screened against these pharmacophores, followed by docking. Biological testing of the 30 top-ranked hits revealed 13 actives, with the best compound showing an IC50 value in the sub-&mu, M range.

Supplemental material, Supplemental_Material for OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics by Lorena Pochini, Michele Galluccio, Mariafrancesca Scalise, Lara Console and Cesare Indiveri in SLAS Discovery

The effect of SH reagents on the human mitochondrial ornithine/citrulline carrier (hORC) was studied. Site directed Cys mutants were employed to gain information on structure/function relationships. The substitutions of each Cys by Ala did not alter the hORC activity measured as [H-3]ornithine/ornithine antiport in proteoliposomes. N ethylmaleimide inhibited the transport of WT with IC50 of 149 mu M. C51A, C50A and C132A showed a much higher IC50. MTSEA and MTSET also inhibited the WT with IC50 of 0.40 mu M and 1.60 mu M, respectively. C51A and C132A showed much higher 1050 values for both reagents. The triple mutant C50/51/132A showed an IC50 for the three reagents that was higher than that of the single mutants. The data strongly suggests that C132, C50 and C51 are involved in inhibition of hORC. Inhibition of WT and mutants by CuPhenanthroline, an S-S forming reagent, suggested that C132 may form disulfides with C50 or C51, impairing the transporter function. The structure/function relationships information deriving from the inhibition studies, were corroborated by the homology structural model of the transporter. The effect of HgCl2 and methyl mercury was also tested on hORC in the light of their capacity to bind thiol residues. Both reagents potently inhibit the transporter. (C) 2018 Elsevier B.V. All rights reserved.

Supplemental material, Supplemental_Material for OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics by Lorena Pochini, Michele Galluccio, Mariafrancesca Scalise, Lara Console and Cesare Indiveri in SLAS Discovery