Nuclear receptors such as the pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are xenobiotic receptors regulating not only drug metabolism and disposition but also various human diseases such as cancer, diabetes, inflammatory disease, metabolic disease and liver diseases, suggesting that PXR and CAR are promising targets for drug discovery. Consequently, there is an urgent need to discover and develop small molecules that target these PXR- and/or CAR-mediated human-disease-related pathways for relevant therapeutic applications. This review proposes approaches to target PXR and CAR, either individually or simultaneously, in the context of various human diseases, taking into consideration the structural differences between PXR and CAR.
PDZ domains are one of the most ubiquitous protein-protein interaction modules found in living systems. Glutaminase interacting protein (GIP), also known as Tax interacting protein 1 (TIP-1), is a PDZ domain-containing protein, which plays pivotal roles in many aspects of cellular signaling, protein scaffolding and modulation of tumor growth. We report here the overexpression, efficient refolding, single-step purification, and biophysical characterization of recombinant human GIP with three different C-terminal target protein recognition sequence motifs by CD, fluorescence, and high-resolution solution NMR methods. It is clear from our NMR analysis that GIP contains 2 alpha-helices and 6 beta-strands. The three target protein C-terminal recognition motifs employed in our interaction studies are glutaminase, beta-catenin and FAS. This is the first report of GIP recognition of the cell surface protein FAS, which belongs to the tumor necrosis factor (TNF) receptor family and mediates cell apoptosis. The dissociation constant ( K D) values for the binding of GIP with different interacting partners as measured by fluorescence spectroscopy range from 1.66 to 2.64 microM. Significant chemical shift perturbations were observed upon titration of GIP with above three ligands as monitored by 2D {(1)H, (15)N}-HSQC NMR spectroscopy. GIP undergoes a conformational change upon ligand binding.
The Glutaminase Interacting Protein (GIP) is composed of a single PDZ domain that interacts with a growing list of partner proteins, including Glutaminase L, that are involved in a number of cell signaling and cancer pathways. Therefore, GIP makes a good target for structure-based drug design. Here we report the solution structures of both free GIP and GIP bound to the C-terminal peptide analog of Glutaminase L. This is the first reported NMR structure of GIP in a complex with one of its binding partners. Our analysis of both free GIP and GIP complexed with the Glutaminase L peptide provides important insights into how a promiscuous binding domain can have affinity for multiple binding partners. Through a detailed chemical shift perturbation analysis and backbone dynamics studies, we demonstrate here that the binding of the Glutaminase L peptide to GIP is an allosteric event. Taken together, the insights reported here lay the groundwork for the future development of a specific inhibitor for GIP.
The
90 kD heat shock proteins (Hsp90) are molecular chaperones
that are responsible for the folding of select proteins, many of which
are directly associated with cancer progression. Consequently, inhibition
of the Hsp90 protein folding machinery results in a combinatorial
attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors
of Hsp90 have entered clinical trials for the treatment of cancer,
all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms, which may lead
to adverse effects. The development of Hsp90 isoform-selective inhibitors
represents an alternative approach toward the treatment of cancer
and may limit some of these detriments. Described herein, is a structure-based
approach to develop isoform-selective inhibitors of Hsp90β,
which induces the degradation of select Hsp90 clients without concomitant
induction of Hsp90 levels. Together, these initial studies support
the development of Hsp90β-selective inhibitors as a method for
overcoming the detriments associated with pan-inhibition.
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