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JOURNAL ARTICLE

Designing a small molecule erythropoietin mimetic

Frank Guarnieri
Methods in Molecular Biology 2015, 1289: 185-210
25709041
Erythropoietin (EPO) is a protein made by the kidneys in response to low red blood cell count that is secreted into the bloodstream and binds to a receptor on hematopoietic stem cells in the bone marrow inducing them to become new red blood cells. EPO made with recombinant DNA technology was brought to market in the 1980s to treat anemia caused by kidney disease and cancer chemotherapy. Because EPO infusion was able to replace blood transfusions in many cases, it rapidly became a multibillion dollar per year drug and as the first biologic created with recombinant technology it launched the biotech industry. For many years intense research was focused on creating a small molecule orally available EPO mimetic. The Robert Wood Johnson (RWJ) group seemed to definitively establish that only large peptides with a minimum of 60 residues could replace EPO, as anything less was not a full agonist. An intense study of the published work led me to hypothesize that the size of the mimetic is not the real issue, but the symmetry making and breaking of the EPO receptor induced by the ligand is the key to activating the stem cells. This analysis meant that residues in the binding site of the receptor deemed absolutely essential for ligand binding and activation from mutagenesis experiments, were probably not really that important. My fundamental hypotheses were: (a) the symmetric state of the homodimeric receptor is the most stable state and thus must be the off-state, (b) a highly localized binding site exists at a pivot point where the two halves of the receptor meet, (c) small molecules can be created that have high potency for this site that will be competitive with EPO and thus can displace the protein-protein interaction, (d) small symmetric molecules will stabilize the symmetric off-state of the receptor, and (e) a key asymmetry in the small molecule will stabilize a mirror image asymmetry in the receptor resulting in the stabilization of the on-state and proliferation of the stem cells into red blood cells. Researchers at Amgen published a co-crystal structure of EPO bound to the EPO receptor, which has a beautiful twofold symmetry-it was argued that this is the active state of the receptor. Activating the EPO receptor with EPO induces an almost instantaneous shutdown mechanism to sharply curtail any proliferative signal transduction, and thus, my hypotheses lead to the conclusion that the Amgen co-crystal is actually the state after receptor downregulation and thus an off-state. To put these hypotheses to the test, my computational method of Simulated Annealing of Chemical Potential was run using the co-crystal created at RWJ, which is the receptor trapped in a partial agonist state. The simulations predicted a previously unknown high affinity binding site at the pivot point where the two halves of the dimeric receptor meet, and detailed analysis of the fragment patterns led to the prediction of a molecule less than 300 MW that is basically twofold symmetric with a chiral center on one side and not the other. Thus, to the degree that computer simulations can be taken seriously, these results support my hypotheses on small molecule receptor activation. When this small molecule was synthesized and tested it indeed induced human hematopoietic stems cells to become red blood cells. When the predicted chiral center of this molecule was removed eliminating its one asymmetric feature, the resulting molecule was an antagonist-it could potently displace hot EPO but could no longer induce stem cell proliferation and differentiation. These results provided strong support for my theories on how to create potent small molecule EPO agonists and were used to launch the new company Locus Pharmaceuticals. These molecules, however, required significant chemical changes in order to make them stable in other in vitro assays and to be in vivo active, but these alterations had to be done in a way that maintained the symmetry-asymmetry considerations that led to the creation of an in vitro active molecule. The combination of changing functional groups to enable good pharmacokinetics, while not changing the key intrinsic symmetry properties were never seriously pursued at Locus and the program died. Investigations into how red blood cells are created have occupied many prominent researchers for the entire twentieth century. In the second half of the century EPO was discovered and by the end of the century it became a blockbuster commercial product that launched the biotech revolution.

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