Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 2. Intracellular analysis of repressor pairs simulating Hdm2–p53 and Hdmx–p53 interactions in the E. coli reporter strain. (A and B) Growth analysis through droplet inoculation of serially diluted strains (10–106 colony forming units (cfu)) expressing the Hdmx–p53 pair of DBD fusions (row 1), the Hmd2–p53 pair of DBD fusions (row 2), a constitutively active repressor control (row 3) (Ref. 22) and an unrepressed (DBDs only) control (row 4) on a (A) non-selective inducing medium (LB with 100 lM IPTG) and (B) selective inducing medium (LB with 100 lM IPTG and 25 lg/mL kanamycin). (C) Reporter-gene (b-galactosidase) activity analysis of Hdm2–p53 and Hdmx–p53 strains. The arrow indicates the expression level (32 lM) for achieving approximately 90% reduction in the reporter expression to be used in the selection procedure.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Expressing, Control, Activity Assay, Selection

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 3. SDS–PAGE analysis of the affinity capture–elution assay performed with the selected SICLOPPS hits. The unspliced constructs containing sequences CIFYYV and CDLRWF were immobilized via chitin-binding domain (CBD) fusion fragments on chitin beads and incubated with an equimolar mixture of Hdm2 or Hdmx (1 mM). Retained materials were subsequently treated with a solution (1 mM) of a p53-derived peptide (ETFSDLWKLL), and the eluate as well as other components of the assay were analyzed by SDS–PAGE. The lane assignments are as follows: lane 1 is a protein ladder; lanes 2 and 3 correspond to the CIFYYV and CDLRWF SICLOPPS constructs, respectively, isolated by chitin beads from crude overexpression lysates; lanes 4 and 5 contain purified Hdm2 and Hdmx, respectively; lane 6 corresponds to an equimolar mixture of Hdm2 and Hdmx; lanes 7 and 8 contain protein material eluted with the p53-derived peptide from the affinity supports containing CIFYYV and CDLRWF leads, respectively, which were pretreated with the equimolar mixture of Hdm2 and Hdm; lanes 9 and 10 were loaded with the post-elution material retained by the chitin beads pre-functionalized with the CIFYYV and CDLRWF constructs, respectively.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: SDS Page, Construct, Binding Assay, Incubation, Derivative Assay, Isolation, Over Expression

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 4. Performance of the selected SICLOPPS constructs and the corresponding single-alanine mutants in the reporter-gene and growth rate assays. (A and B) ONPG assay data and droplet inoculation analysis, respectively, of the anti-Hdm2 CIFYYV construct and its mutants in the Hdm2–p53 strain. (C and D) ONPG assay data and droplet inoculation analysis, respectively, of CDLRWF and its mutants in the Hdmx–p53 strain.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Construct

Journal: Bioorganic & Medicinal Chemistry

Article Title: Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system

doi: 10.1016/j.bmc.2010.06.053

Figure Lengend Snippet: Figure 5. Surface representations of Hdm2 (PDB: 1YCR; left) and Hdmx (PDB: 3DAB; right) in bound states with ligands (p53 residues 15–29) not shown for clarity. The proteins are colored according to the elemental make-up (C, gray; O, red; N, blue; S, yellow). The p53-binding pockets with labeled Leu, Trp and Phe subsites (green) are outlined to highlight the topological differences in the respective binding pockets. The residues proposed to be responsible for differences in ligand recognition patterns (F86 and H96 in Hdm2; L85 and P95 in Hdmx) are indicated by arrows.

Article Snippet: Linear p53 peptide ETFSDLWKLL was synthesized by ChemImpex, Inc., in 95% HPLC purity.

Techniques: Binding Assay, Labeling

Journal: bioRxiv

Article Title: A small molecule PTER-selective inhibitor reduces food intake and body weight

doi: 10.64898/2026.01.26.701829

Figure Lengend Snippet: (A) Superposition of human HDAC(1-11) structures showing conserved aromatic side chains in active site. Average distance between two side chain is labeled. PDB IDs are 4BKX, 4LXZ, 4A69, 2VQJ, 5EDU, 3C0Y, 1T64 for human HDAC 1-4, 6-8. HDAC5 and 9-11 are AlphaFold-predicted models. The representative SAHA (white) is from an HDAC2 co-crystal structure (PDB ID 4LXZ). (B) Superposition of the top poses of docked PTERi (yellow) in sPTER and docked SAHA in sPTER, and SAHA from an HDAC2 co-crystal structure (PDB ID 4LXZ). Superposition is based on the top-ranked SAHA pose in sPTER and SAHA pose in HDAC2. SAHA poses are not shown for simplicity. Side chains of sPTER are shown in purple, while side chains of HDAC2 are shown in white. (C) Chemical structure of PTERi. (D) Dose-response inhibition of PTER activity by PTERi. (E) Heat map of dose-response inhibition for PTERi against the indicated recombinant enzyme. (F, G) Lineweaver-Burke plot (F) and dose-response inhibition of PTERi (G) in PTER activity assays. For (D-G) , PTER activity (N-acetyltaurine hydrolysis) was measured by quantifying taurine production using 200 ng of purified recombinant mouse PTER (mPTER, panels D-G ) or purified recombinant PTER from the indicated species (G) and 100 µM N-acetyltaurine for 1 h at 37°C. N=3/data point for (D,G) and N=1/data point for (E,F) . (D,G) are shown as mean ± SEM. IC 50 values for were determined from the dose-response curves via nonlinear regression analysis using GraphPad Prism.

Article Snippet: The HDAC assay was performed in 50 μl reaction volume containing the HDAC reaction assay buffer, 600 μg of the liver lysate, 100 μM HDAC substrate peptide Ac-Arg-Gly-Lys-Ac-Glu-AMC (custom synthesized by Elim Biopharm), and 10 μM of indicated inhibitors.

Techniques: Labeling, Inhibition, Activity Assay, Recombinant, Purification