Aimée Martin

Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1.

We produced the human leukotriene B(4) (LTB(4)) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% alpha-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB(4) with K(a)=7.8(+/-0.2)x10(8)M(-1) and a stoichiometric ratio of 0.98(+/-0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB(4). We report evidence that both partners, LTB(4) and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB(4) conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.

G Protein Activation by the Leukotriene B4 Receptor Dimer EVIDENCE FOR AN ABSENCE OF TRANS-ACTIVATION

There is compelling evidence that G protein-coupled receptors exist as homo- and heterodimers, but the way these assemblies function at the molecular level remains unclear. We used here the purified leukotriene B4 receptor BLT1 stabilized in its dimeric state to analyze how a receptor dimer activates G proteins. For this, we produced heterodimers between the wild-type BLT1 and a BLT1/ALXR chimera. The latter is no longer activated by leukotriene B4 but is still activated by ALXR agonists. In this heterodimer, agonist binding to either one of the two protomers induced asymmetric conformational changes within the receptor dimer. Of importance, no G protein activation was observed when using a dimer where the ligand-loaded protomer was not able to trigger GDP/GTP exchange due to specific mutations in its third intracellular loop, establishing that the conformation of the agonist-free protomer is not competent for G protein activation. Taken together, these data indicate that although ligand binding to one protomer in the heterodimer is associated with cross-conformational changes, a trans-activation mechanism where the ligand-free subunit would trigger GDP/GTP exchange cannot be considered in this case for G protein activation. This observation sheds light into the way GPCR dimers, in particular heterodimers, could activate their cognate G proteins.

Spermidinyl-CoA-based HAT inhibitors block DNA repair and provide cancer-specific chemo- and radiosensitization

Acetyl group turnover on specific lysine ε-amino groups of the core chromosomal histones regulates DNA accessibility function, and the acetylating and deacetylating enzymes that govern the turnover provide important targets for the development of anti-cancer drugs. Histone deacetylase (HDAC) inhibitors have been developed and evaluated extensively in clinical trials, while the development of inhibitors of histone acetyltransferase (HAT) has proceeded more slowly. Here we have examined the cellular effects of an S-substituted coenzyme A (CoA) inhibitor of histone acetylation, consisting of spermidine (Spd) linked to the S-terminus of CoA through a thioglycolic acid linkage (adduct abbreviated as Spd-CoA), as well as the effects of a truncated Spd-CoA derivative lacking the negatively charged portion of the CoA moiety. While exposure of cancer cells to Spd-CoA has little effect on cell viability, it causes a rapid inhibition of histone acetylation that correlates with a transient arrest of DNA synthesis, a transient delay in S-phase progression, and an inhibition of nucleotide excision repair and DNA double strand break repair. These effects correlate with increased cellular sensitivity to the DNA-targeted chemotherapeutic drugs, cisplatin (Platinol™) and 5-Fluorouracil, to the DNA damaging drug, camptothecin, and to UV-C irradiation. The sensitization effects of Spd-CoA are not observed in normal cells due to a barrier to uptake. The truncated Spd-CoA derivative displays similar but enhanced chemosensitization effects, suggesting that further modifications of the Spd-CoA structure could further improve potency. The results demonstrate that Spd-CoA and its truncated version are efficiently and selectively internalized into cancer cells, and suggest that the resulting inhibition of acetylation-dependent DNA repair enhances cellular sensitivity to DNA damage. These and related inhibitors of histone acetylation could therefore constitute a novel class of potent therapy sensitizers applicable to a broad range of conventional cancer treatments.

Engineering a G protein-coupled receptor for structural studies: Stabilization of the BLT1 receptor ground state

Structural characterization of membrane proteins is hampered by their instability in detergent solutions. We modified here a G protein‐coupled receptor, the BLT1 receptor of leukotriene B4, to stabilize it in vitro. For this, we introduced a metal‐binding site connecting the third and sixth transmembrane domains of the receptor. This modification was intended to restrain the activation‐associated relative movement of these helices that results in a less stable packing in the isolated receptor. The modified receptor binds its agonist with low‐affinity and can no longer trigger G protein activation, indicating that it is stabilized in its ground state conformation. Of importance, the modified BLT1 receptor displays an increased temperature‐, detergent‐, and time‐dependent stability compared with the wild‐type receptor. These data indicate that stabilizing the ground state of this GPCR by limiting the activation‐associated movements of the transmembrane helices is a way to increase its stability in detergent solutions; this could represent a forward step on the way of its crystallization.

New tensio-active molecules stabilize a human G protein-coupled receptor in solution

Structural characterization of membrane proteins is hampered by the instability of the isolated proteins in detergent solutions. Here, we describe a new class of phospholipid‐like surfactants that stabilize the G protein‐coupled receptor, BLT1. These compounds, called C13U9, C13U19, C15U25 and C17U16, were synthesized by radical polymerization of Tris(hydroxymethyl) acrylamidomethane in the presence of thioglycerol, first endowed with two hydrocarbon chains with variable lengths (13–17 carbon atoms), as transfer reagent. C13U19, C17U16 or C15U25 significantly enhanced the stability of BLT1 in solution compared to what was obtained with common detergents. These molecules therefore represent a promising step towards the structural characterization of BLT1 and possibly other membrane proteins.

Regioselective synthesis of inhibitors of histone acetyl transferase covalently linking spermidine to the s-terminus of coenzyme a and fragments.

Abstract The reaction of a bromoacetylthioester BrCH2CO-S-R (R radical in the coenzyme A series) with spermidine (Spd) derivatives is investigated and it is established that the adduct SpdCOCH2-S-R 1 is the product of the reaction. Parallel studies with model compounds show that this is a general reaction of bromoacetylthioesters. The synthesis of analogs of 1 is described and they correspond to inhibitors of the histone acetyltransferase.

Abstract 5444: Polyamine-based HAT inhibitors as therapy sensitizers

Histone acetylation plays an important role in regulating chromatin structure and is tightly controlled through the actions of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulation of either HAT or HDAC activity can affect chromatin remodeling needed to carry out its functions and contribute to cancer. While HATs are potential targets for anticancer therapeutics, the development of effective HAT inhibitors has been slow. We have examined the cellular effects of an S-substituted coenzyme A (CoA) inhibitor of histone acetylation, consisting of spermidine (Spd) linked to the S-terminus of CoA through a thioglycolic acid linkage (adduct abbreviated as Spd-CoA), as well as the effects of a truncated Spd-CoA derivative lacking the negatively charged portion of the CoA moiety. While exposure of cancer cells to Spd-CoA has little effect on cell viability, it causes a rapid inhibition of specific acetylated lysines, H3-K9 and H3-K56. That inhibition correlates with a transient arrest of DNA synthesis, a transient delay in S-phase progression, and an inhibition of nucleotide excision repair and DNA double strand break repair. Furthermore, Spd-CoA synergizes with a variety of commonly used DNA damaging chemotherapeutic agents, cisplatin (Platinol TM ) and 5-fluorouracil, camptothecin, as well as UV-C radiation to induce cancer cell killing. This suggests that a common mechanism, relevant to DNA damage, underlies the ability of histone acetylation inhibition to synergize with drugs and radiation. Normal human fibroblasts and epithelial cells are not sensitized to DNA damage by Spd-CoA due to a barrier to uptake, indicating that this differential uptake can be exploited to achieve cancer cell-specific sensitization. Furthermore, therapy sensitization occurs in both p53-null cancer cells and in cancer cells expressing wild-type p53, indicating that p53-mediated apoptosis is not required. The truncated Spd-CoA derivative displays similar but enhanced chemosensitization effects, suggesting that this class of inhibitors may be amenable to further refinement and have considerable clinical potential as a novel class of potent therapy sensitizers applicable to a broad range of conventional cancer treatments, particularly to reduce therapy toxicity and reverse therapy resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5444.