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Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel.

ABSTRACT: We obtained unanticipated synthetic byproducts from alkylation of the ?(1) nitrogen (N3) of the histidine imidazole ring of the polo-like kinase-1 (Plk1) polo-box domain (PBD)-binding peptide PLHSpT. For the highest-affinity byproduct, bearing a C(6)H(5)(CH(2))(8)- group, a Plk1 PBD cocrystal structure revealed a new binding channel that had previously been occluded. An N-terminal PEGylated version of this peptide containing a hydrolytically stable phosphothreonyl residue (pT) bound the Plk1 PBD with affinity equal to that of the non-PEGylated parent but showed markedly less interaction with the PBDs of the two closely related proteins Plk2 and Plk3. Treatment of cultured cells with this PEGylated peptide resulted in delocalization of Plk1 from centrosomes and kinetochores and in chromosome misalignment that effectively induced mitotic block and apoptotic cell death. This work provides insights that might advance efforts to develop Plk1 PBD-binding inhibitors as potential Plk1-specific anticancer agents.

SUBMITTER: Liu F

PROVIDER: S-EPMC3158281 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel.

Liu Fa F Park Jung-Eun JE Qian Wen-Jian WJ Lim Dan D Gräber Martin M Berg Thorsten T Yaffe Michael B MB Lee Kyung S KS Burke Terrence R TR

Nature chemical biology 20110717 9

We obtained unanticipated synthetic byproducts from alkylation of the δ(1) nitrogen (N3) of the histidine imidazole ring of the polo-like kinase-1 (Plk1) polo-box domain (PBD)-binding peptide PLHSpT. For the highest-affinity byproduct, bearing a C(6)H(5)(CH(2))(8)- group, a Plk1 PBD cocrystal structure revealed a new binding channel that had previously been occluded. An N-terminal PEGylated version of this peptide containing a hydrolytically stable phosphothreonyl residue (pT) bound the Plk1 PBD ...[more]

PMID: 21765407

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Project description:The human hepatic organic cation transporter 1 (hOCT1) is a well-known transporter of both xenobiotic and endogenous cations. The substrates and inhibitors of hOCT1 are structurally and physiochemically diverse and include some widely prescribed drugs (metformin and imatinib), vitamins (thiamine), and neurotransmitters (serotonin). It has been demonstrated that the closely related renal isoform, hOCT2, is subject to ligand-dependent modulation, wherein one ligand may enhance or inhibit transport of a second, chemically unrelated, ligand. This phenomenon has important implications for drug-drug interactions due to the ubiquity of polypharmacy and the large number of drugs that are present as cations under physiological conditions. Therefore, the objective of this study was to determine if hOCT1 is subject to the same ligand-dependent modulation as hOCT2, and to identify unique putative ligand binding sites in the translocation channel for a sub-set of ligands using computational modeling. The competitive counter flow (CCF) assay was employed to examine ligand-dependent effects by utilizing four different radiolabeled probe substrates: MPP+, serotonin, metformin, and TEA. We identified 20 ligands that modulated the transport of the four test substrates examined. One of the putative ligands identified, BSP, is an anion at physiological pH. Direct uptake studies of radiolabeled BSP suggested that it is a hOCT1 substrate with a Km of 13.6?±?2.6?µM and Vmax of 55.1?±?4.1?pmol/mg protein/min. Each ligand identified was computationally docked into a homology model of hOCT1 using the UCSF DOCK software package. The docking study revealed three separate ligand binding pockets within the hOCT1 translocation pathway, defined by their interactions with three prototypical substrates: MPP+, TEA, and acyclovir. Our results suggest that hOCT1 is not only subject to ligand-dependent modulation, but also that individual ligand binding occurs at discrete sites within the hOCT1 translocation pathway which may influence ligand binding at the other sites.

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Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel.

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Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel.

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