Research

  1. Dynamics of Ras proteins in solution and in membrane






  2. Dynamics of Ras proteins in solution and in membrane

    Simulations have played a key role in elucidating the dynamics of Ras proteins in the aqueous and membrane environments. We use classical and advanced molecular dynamics simulations to study the isolated catalytic domain in water, the lipid-anchor and the full-length Ras in bilayers of various lipid composition. Our current focus is on K-Ras, which is the most frequently mutated Ras isoform in human cancers and developmental disorders. Ongoing projects revolve around the question of how somatic and genetic mutations may alter the population of conformational states and oligomerization behavior of K-Ras, and how these may affect interaction with effectors or modulators.



    Selected Publications

    1. Prakash P and Gorfe AA, "Lessons from computer simulations of Ras proteins in solution and in membrane", BBA-General Subjects, 2013, 1830(110): 5211-5218.
    2. Prakash P, Sayyed-Ahmad A and Gorfe AA,"The Role of Conserved Waters in Conformational Transitions of Q61H K-ras", PLoS Computational Biology, 2012, 8(2), e1002394.
    3. Janosi L and Gorfe AA, "Segregation of negatively charged phospholipids by the polycationic and farnesylated membrane anchor of Kras", Biophysical Journal, 2010, 99:3666-3674.
    4. Gorfe AA, Hanzal-Bayer M, Abankwa D, Hancock JF and McCammon JA, "Structure and dynamics of the full-length lipid-modified H-ras protein in complex with a DMPC bilayer", Journal of Medicinal Chemistry, 2007, 50:674-84
  3. Organization of Ras proteins in membrane domains






  4. Organization of Ras proteins in membrane domains

    Our work on membrane-organization of Ras proteins involves studying various lipid bilayer models using atomically detailed and coarse-grained simulations of Ras-membrane complexes. Our previous studies led to important insights into the physical basis for clustering and non-overlapping distribution of different Ras proteins in membrane domains. In particular, we found that the nature of lipid-modification dictates the distinct lateral organization of different Ras proteins on the plasma membrane, and that cholesterol modulates lipid domain stability and thereby the stability of Ras nanoclusters. We also found that surface-bound proteo-oligomers can be used to probe the mechanism by which membrane curvature might be generated and/or maintained. However, a number of technical challenges remain to be solved in order to accurately and fully model oligomers of surface-bound Ras and other lipid-modified proteins, which is the object of our current focus.

    Selected Publications

    1. Janosi L and Gorfe AA, "Simulating POPC and POPC/POPG bilayers: Conserved packing and altered surface reactivity", Journal of Chemical Theory and Computation, 2010, 16:3267-3273.
    2. Janosi L and Gorfe AA, "Importance of the sphingosine base double bond geometry for the structural and thermodynamic properties of sphingomyelin bilayers(Featured Article)", Biophysical Journal, 2010, 99 (9):2957–2966.
    3. Janosi L, Li Z, Hancock JF and Gorfe AA, "Organization, Dynamics and Segregation of Ras Nanoclusters in Membrane Domains", Proceedings of the National Academy of Sciences USA, 2012, 109(21): 8097-102.
    4. Li Z, Janosi L and Gorfe AA, "Formation and Domain-partitioning of H-ras Peptide Nanoclusters: Effects of Peptide Concentration and Lipid Composition", Journal of the American Chemical Society, 2012, 134(41):17278-85.
  5. Targeting an illusive foe






  6. Targeting an illusive foe

    As part of a broader effort to developing anti-Ras therapeutics, we leverage insights emerging from the projects described above for the design of inhibitors that directly act on Ras. Our previous studies provided the initial clues about the allosteric nature of Ras and the role of conformational selection in its function. This led us to contemplate the potential druggability of Ras at the time when this was thought hopeless. Spurred in part by findings from large-scale genomic studies that the KRAS gene remains to be the main culprit in many forms of cancer, Ras is now back in the forefront of the search for new anti-cancer therapeutics. Our contribution to this effort includes the identification of novel allosteric ligand binding sites and prediction of small-molecule ligands that might bind to these sites. Moreover, working with cell biologists and pharmacologists, we showed for the first time that nucleotide exchange factors are required for oncogenic Ras signaling and inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras. Our current effort in the search for isoform-selective Ras inhibitors includes developing methods to incorporate membrane into our dynamics-guided, ensembled-based drug-design scheme.

    Selected Publications

    1. Gorfe AA, "Mechanisms of allostery and membrane attachment in Ras GTPases: Implications for anti-cancer drug discovery", Current Medicinal Chemistry, 2010, 17, 1-9.
    2. Hocker JH, Kwang-Jin C, Chung-Ying KC, Rambahal N, Sagineedu SR, Shaari K, Stanslas J, Hancock JF, Gorfe AA, "Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function", Proceedings of the National Academy of Sciences USA, 2013, 110(25):10201-10206.
    3. Grant JN, McCammon JA and Gorfe AA, "Conformational Selection in G-proteins: Lessons from Ras and Rho(Featured Article)", Biophysical Journal, 2010, 99:L87-L89.
    4. Grant BJ, Lukman S, Hocker H, Sayyah J, Heller Brown J, McCammon JA, Gorfe AA,"Novel Allosteric Sites on Ras for Lead Generation", PLoS ONE, 2011, 6, e25711.
    5. Prakash P, Sayyed-Ahmad A and Gorfe AA,"The Role of Conserved Waters in Conformational Transitions of Q61H K-ras", PLoS Computational Biology, 2012, 8(2), e1002394.
  7. Interaction of NSAIDs with micelles and bilayers






  8. Interaction of NSAIDs with micelles and bilayers

    We would like to understand the physical basis for the ability of common non-steroidal anti-inflamatory drugs (NSAIDs), such as aspirin, to cause gastrointestinal injury through their detrimental effect on surface membranes. This effect appears to be exacerbated by bile salts and reduced or eliminated by conjugating NSAIDs with phosphatidylcholines (PCs). We study the surface behavior, morphology and size of micellar particles that appear during simulations of binary and ternary mixtures of NSAIDs and bile salts with different PCs. Initial results suggest that NSAIDs can form potentially cytotoxic aggregates with bile acids, and may alter the normal physiologic balance between bile acids and PCs .



    Selected Publications

    1. Prakash P and Gorfe AA, "Phosphatidylcholine attenuates aggregation of non-steroidal anti-inflammatory drugs with bile acids", Biochemistry, 2013, 52(42): 7461–7469.
    2. Prakash P, Sayyed-Ahmad A, Zhou Y, Volk DE, Gorenstein DG, Dial EJ, Lichtenberger LM and Gorfe AA, "Aggregation Behavior of Ibuprofen, Cholic Acid and Dodecylphosphocholine Micelles", BBA-Biomembranes, 2012, 1818(12): 340-347.
    3. Sayyed-Ahmad A, Lichtenberger LM and Gorfe AA, "Structure and Dynamics of Cholic Acid and Dodecylphosphocholine−Cholic Acid Aggregates", Langmuir, 2010, 26:13407-13414.
  9. Methods Development






  10. Methods Development

    We support all of our computational studies by the development of novel computational methods. These include techniques to expedite binding site identification and docking to ensembles of protein conformations both in solution and membrane environments. For instance, our method LIBSA analyzes multiple docked poses against a single or ensemble of receptor conformations and returns a metric for the relative binding to a specific region of interest. By using novel filtering algorithms and the signal-to-noise ratio, the relative ligand-binding frequency at different pockets can be calculated and compared quantitatively. Ligands can then be triaged by their tendency to bind to a site instead of ranking by affinity alone. Our most recent method, pMD-membrane, allows for the powerful probe-based molecular dynamics approach for the identification of druggable sites to be applicable to membrane-bound targets. pMD-membrane overcomes the negative impact of probes on bilayer structure by re-parameterizing selected pairwise interactions between probes and bilayer lipids. We have also developed a number of analysis tools that facilitate analysis of clusters of surface-bound proteins.

    Selected Publications

    1. Hocker HJ, Maharaj N and Gorfe AA, "LIBSA - Method for the determination of ligand-binding preference to allosteric sites on receptor ensembles", Journal of Chemical Information and Modeling, 2014, 24;54(2):530-538.
    2. Prakash P, Sayyed-Ahmad A and Gorfe AA, "pMD-membrane: A method for ligand binding site identification in membrane-bound proteins", PLoS Comput Biol 2015, In Press.
    3. Prakash P, Hancock JF and Gorfe AA, "Binding hotspots on K-Ras: Consensus ligand binding sites and other reactive regions from probe-based molecular dynamics analysis", Proteins 2015, 83(5): 898-909.
    4. Janosi L, Li Z, Hancock JF and Gorfe AA, "Organization, Dynamics and Segregation of Ras Nanoclusters in Membrane Domains", Proceedings of the National Academy of Sciences USA, 2012, 109(21): 8097-102.








    Downloads

    1. Download LIBSA Code
    2. Download Organization, Dynamics and Segregation of Ras Nanoclusters in Membrane Domains Supplementary