Sickle cell disease molecular pathogenesis and drug therapy

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

1. Screening for new drugs. Although the molecular target for drug therapy of sickle cell disease has been known since Linus Pauling's discovery that sickle cell anemia is a molecular disease over 70 years ago, only 2 ant-sickling drugs have been approved by the United States Food and Drug Administration. One is hydroxyurea. Hydroxyurea reduces chronic organ damage and the frequency of pain crises. It does so by increasing the synthesis of fetal hemoglobin, which dilutes the abnormal hemoglobin S, markedly slowing its polymerization to form the fibers that distort (sickle) and make the red cells inflexible. This drug is, however, only partially successful in reducing the frequency of pain crises and the chronic organ damage characteristic of the disease. A second, controversial drug, voxelotor, was approved in November 2019 by the FDA. It acts by preferentially binding to the R quaternary conformation, which cannot polymerize. There is a modest increase in hemoglobin levels for patients taking this drug, but there is no evidence yet that it decreases organ damage or reduces the frequency of pain crises. We have performed detailed sickling simulations that show voxelotor actually increases anemia, because hemoglobin with molecules bound mare stuck in the high affinity R conformation and deliver no oxygen. The search for additional and more effective therapeutic agents has been severely hampered by the lack of a rapid and sensitive assay for inhibition of sickling. A truly high throughput screen has been developed using a sophisticated automated microscope system (Biotek Lionheart) based on nitrogen deoxygenation of red cells in a 384 well plate format and a robust machine learning algorithm to determine the time at which an individual cell sickles. The assay has been used to screen the 12,657 compound library of compounds that have been tested in humans from the Scripps Institute funded by the Gates Foundation at 10 micromolar. Inhibitors from this screen were investigated at 9 concentrations from 1 nanomolar to 10 micromolar to determine the concentrations at which they are effective. So far, we have discovered 105 compounds from this library that inhibit sickling to a degree that is potentially therapeutic. Once we know the concentrations of these "hits" that are found in the serum of humans, information that has been challenging to determine, we can determine which compounds are candidates should be further investigated in the detail that is necessary to begin clinical trials. To compare with serum concentrations will require measurements on whole blood instead of the highly diluted blood (1:1000) used in the assay. One of our hits is the common spice curcumin. However, it is ineffective as an oral drug because it is completely metabolized by the liver to derivatives that show no inhibition of sickling. Wed are now collaboeating with groups outside NIH to deliver the drug transdermally. Our sickling assay turns out to be a robust biomarker of clinical severity and is being used to evaluate sickling in blood from patients on gene thereapy or stem cell transplantation. In a parallel, project measurements of oxygen binding and cell volume are being made to determine whether the mechanism of anti-sickling is switching the quaternary structure from the polymerizing T conformation to the non-polymerizing R conformation or from the decrease in intracellular hemoglobin concentration from an increase in cell volume. Knowing the mechanism will inform us on what drugs can be taken together because they do not compete for the same target. Clinical trials with Mitapivat. Increased intracellular 2,3- diphosphoglycerate (2,3-DPG) stabilizes fibers and promotes sickling, while decreased intracellular adenosine triphosphate (ATP) levels can lead to hemolysis. Mitapivat (AG-348) is an oral, small molecule, allosteric activator capable of activating both mutant and wild type red cell pyruvate kinase (PKR), thus decreasing 2,3-DPG and increasing ATP levels in red cells and potentially acting as an anti-sickling agent. The safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of multiple ascending doses of mitapivat were assessed in subjects with SCD in a Phase I study. Mitapivat demonstrated an acceptable safety profile across the tested dose levels in 8 subjects with SCD. Analyses of data show promising evidence of efficacy in terms of Hb increase from baseline with concomitant decreases in hemolytic markers. The accompanying changes in metabolites and sickling studies are consistent with the proposed mechanism of the drug. The study is ongoing with a planned sample size of 15 subjects in a phase 2/3 clinical trial directed by Dr. Swee Lay Thein (NHLBI).