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The Bh3 Domain of Puma:Structure Determination and Small
Molecular Inhibitor Design
Nicki Zevola
Bahar Lab
Lab Meeting March 25, 2009
Intro. What are BCl-2 proteins?• A class of pro-survival and
pro-apoptotic proteins– Easily distinguishable– Pro-survival: BclXL, Bcl-w, MCl-
1, A1, Bax, Bak• 2-4 BH domains
– Pro-apoptotic: Bim, Bad, Bmf, Bid, Bik, Hrk, Noxa, Puma
• Only 1 BH domain, Bh3
• Key interactions occur when pro-apoptotic proteins (e.g., Puma) inhibit pro-survival proteins (e.g., BclXL)
Crystal structure of BCl-xl (mouse)
Crystal structure of Puma (mouse)
1
3
2
1
Project Overview• Goal of Project: Develop small
molecule mitochondrial-targeted drugs to inhibit the binding of the Puma Bh3 domain to Bcl-2 family proteins.
• Normally, Puma Bh3 domain:Bcl-2 protein binding antagonizes anti-apoptotic Bcl-2 proteins, inhibiting Apaf-1, and resulting in caspase release and apoptosis.– Studies with Puma-deficient mice have
shown that Puma not only activates, but is required for, apoptosis induced by oncogenes and DNA-damaging agents.1,2
1Qui et. al. PUMA Regulates Intestinal Progenitor Cell Radiosensitivity and Gastrointestinal Syndrome. Cell Stem Cell. 2008. 2(6):576-583.2 Yu J., Zhang L., Hwang PM, Kinzler K.W., Vogelstein B. Puma induces the rapid apoptosis of colorectal cancer cells. Mol. Cell. 2001. 7:3:673-82.
How We Will Find Small Molecular Inhibitors for Puma (Project Design)
Analyze structure of Bh3 domain of Puma
Determine key conserved interactions between Puma Bh3 domain and Bcl-2 proteins
Develop pharmacophore models using conserved interactions
Screen Zinc 8.0 database for lead-like compounds that match pharmacophore model
Develop Puma models to prioritize hits
In vitro testing
Day et. al.: How Puma Bh3 Domain Bh3-Mcl1 and Noxa Bh3-Mcl1 Structures were Obtained
• 1. Two samples made
– 1. 13C, 15N-labeled peptide + unlabeled Mcl-1
– 2. Unlabeled peptide + labeled Mcl-1
• 2. NMR techniques used for resonance assignments
• 3. Structures evaluated for proper geometry
– No distance violation > 0.2 Å
– No angle violation > 5o
•Why only the Bh3 Domain of Puma, Noxa with Mcl-1?
Puma and Noxa have extremely unstable structures, and hence only the Bh3 binding domain structure has been accurately determined. In the Bahar lab, we have used the i-Tasser server (Zhang) to help ascertain the entire Puma structure (see next slide).
(2rod) (2roc)
i-Tasser• Sequence of hPUMA
submitted to online server developed by Dr. Yang Zhang, Ph.D. (University of Kansas)– Finalist at CASP (Critical
Assessment of Structural Predictions) Competition
• Server uses LOMETS (a form of threading) in structural prediction
• 5 models generated for hPuma – Problem: Best C-score:
-3.46• Will be addressed later
Day et. al.: Comparison of MCl-1 complexes with other BCl-2 proteins
• 1. Superimposition of Mcl-1:Puma with:
• BClXL:Bim, • A1:Puma, • and BclXL
– RMSD of backbone BClXL:Bim and Mcl-1: Puma: 1.5 Å
– Most notable structural difference: BclXLhas a long loop between α1 and α2 not found in Mcl-1 or A1
Day et. al.: Comparison of Mcl-1 complexes with other Bcl-2 proteins (con’t)
• 2. Structural Alignment– Sequence comparison (excluding BclXL loop
residues):• BclXL,Mcl-1: 25.6% identity, 40.0% similarity
• A1, Mcl-1: 25.4% identity, 46.% similarity
Day et. al.: The Consensus Bh3 Domain Binding Motif
• The 13 residue consensus sequence that defines the Bh3 domain in PUMA is: φ1ΣXXφ2XXφ3ΣDZφ4L– where φ1-φ4 are hydrophobic
residues– Σ are small residues– Z is usually an acidic residue– L is a hydrophilic residue
capable of forming an intermolecular cap
– D is the conserved aspartate Bh3 domains of Noxa (in yellow, left) and Puma (in pink, right).
in situ Mutagenesis Reveals Other Key Residues (Yu et. al., 2009)
• Already known from Day et. al.: conserved interactions with Bcl2 proteins
• Jian Yu et. al. (Univ. of Pittsburgh): 2 specific point mutations alter binding specificity upstream of the Bh3 binding domain!– These are therefore included in our pharmacophore model (next step)
Images courtesy Gabriela Mustata, Ph.D.
Pharmacophore Model Development
• Our superimposition of our hPuma-A1 (from i-Tasser) with hPUMA-Mcl1 (from 2roc) structures resulted in an RMSD of 0.55Å!
• Four key interactions of Bcl2 proteins and hPuma (see next slide):– Phe251.CB (Bcl2) – Leu141.CD1 (hPuma)– Ph395.CD1 (Bcl2) – Leu141.CD1 (hPuma)– Asp237.OD2 (Bcl2) – Asp142.NH1 (hPuma)– Arg244.NE (Bcl2) – Asp146.OD1 (hPuma)
Pharmacophore Search Results Thus Far
Zinc 7.0 Lead-like Database• Found 1 hit
Zinc 8.0 Lead-like Database•So far…Screened 24/37 of database (about 1.3 x 106 compounds)
•Found 20 hits thus far (some shown below)
Current Problem: i-Tasser model
• 5 models of hPuma generated by i-Tasser are not entirely satisfactory, as shown by Fast Contact server (Camacho et. al.)
• Current solution: Use MD Simulations for energy minimization of i-Tasser models, recalculate energies on Fast Contact server