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Stem-cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD) but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available to the vast majority of patients suffering from this disease for many years. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only successful drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here we report results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFrame drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFrame library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant anti-sickling at concentrations ranging from 31 nM to 10 μM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 anti-sickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic effect for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle-syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.Significance StatementThe vast majority of patients suffering from sickle cell disease live in under-resourced countries. Consequently, advanced medical facilities required for curative therapies, such as stem cell transplantation and gene therapy, will be unavailable to them for a long time. Hydroxyurea, approved by the FDA in 1998, is the only effective drug that inhibits polymerization of the mutant hemoglobin S that stiffens and distorts (“sickles”) red cells, the root cause of the pathology. What is urgently needed now for these patients are additional, inexpensive oral anti-sickling drugs. Our high throughput phenotypic screen of the ReFrame drug repurposing library reported here discovered 106 compounds that are anti-sickling. On a statistical concentration basis, as many as 21 are predicted to be potential drugs.

The issue of treating sickle cell disease with drugs that increase hemoglobin oxygen affinity has come to the fore with the US Food and Drug Administration approval in 2019 of voxelotor, the only antisickling drug approved since hydroxyurea in 1998. Voxelotor reduces sickling by increasing the concentration of the nonpolymerizing, high oxygen affinity R (oxy) conformation of hemoglobin S (HbS). Treatment of sickle cell patients with voxelotor increases Hb levels and decreases indicators of hemolysis, but with no indication as yet that it reduces the frequency of pain episodes. In this study, we used the allosteric model of Monod, Wyman, and Changeux to simulate whole-blood oxygen dissociation curves and red cell sickling in the absence and presence of voxelotor under the in vivo conditions of rapid oxygen pressure decreases. Our modeling agrees with results of experiments using a new robust assay, which shows the large, expected decrease in sickling from the drug. The modeling indicates, however, that the increase in oxygen delivery from reduced sickling is largely offset by the increase in oxygen affinity. The net result is that the drug increases overall oxygen delivery only at the very lowest oxygen pressures. However, reduction of sickling mitigates red cell damage and explains the observed decrease in hemolysis. More importantly, our modeling of in vivo oxygen dissociation, sickling, and oxygen delivery suggests that drugs that increase fetal Hb or decrease mean corpuscular hemoglobin concentration (MCHC) should be more therapeutically effective than drugs that increase oxygen affinity.

Significance The root cause of pathology in sickle cell disease is the polymerization of the mutant hemoglobin S upon deoxygenation in the tissues to form fibers. Both the amount of fiber at equilibrium and the kinetics of fiber formation depend on the partial pressure of oxygen. We show that control of polymerization by oxygen at equilibrium can be better explained by a recent extension of the famous two-state allosteric model of Monod, Wyman, and Changeux. Because of the unusual kinetics, polymerization is far out of equilibrium, which explains why patients with the disease manage to survive, while those with high levels of fetal hemoglobin and those with sickle trait (the heterozygous condition) have relatively benign conditions., The pathology of sickle cell disease is caused by polymerization of the abnormal hemoglobin S upon deoxygenation in the tissues to form fibers in red cells, causing them to deform and occlude the circulation. Drugs that allosterically shift the quaternary equilibrium from the polymerizing T quaternary structure to the nonpolymerizing R quaternary structure are now being developed. Here we update our understanding on the allosteric control of fiber formation at equilibrium by showing how the simplest extension of the classic quaternary two-state allosteric model of Monod, Wyman, and Changeux to include tertiary conformational changes provides a better quantitative description. We also show that if fiber formation is at equilibrium in vivo, the vast majority of cells in most tissues would contain fibers, indicating that it is unlikely that the disease would be survivable once the nonpolymerizing fetal hemoglobin has been replaced by adult hemoglobin S at about 1 y after birth. Calculations of sickling times, based on a recently discovered universal relation between the delay time prior to fiber formation and supersaturation, show that in vivo fiber formation is very far from equilibrium. Our analysis indicates that patients survive because the delay period allows the majority of cells to escape the small vessels of the tissues before fibers form. The enormous sensitivity of the duration of the delay period to intracellular hemoglobin composition also explains why sickle trait, the heterozygous condition, and the compound heterozygous condition of hemoglobin S with pancellular hereditary persistence of fetal hemoglobin are both relatively benign conditions.

The polymerization of the mutant hemoglobin S upon deoxygenation to form fibers in red blood cells of patients suffering from sickle-cell anemia results in changes in cell shape and rigidity, also known as sickling, which underlie the pathology of the disease. While much has been learned about the fundamental physical chemistry of the polymerization process, transferring these insights to sickling of red cells under in vivo conditions requires being able to monitor, and ultimately to predict, the time course of cellular sickling under physiological conditions of deoxygenation. To this end, we have developed an experimental technique for tracking the temporal evolution of the sickling of red blood cells under laboratory deoxygenation conditions, based on the automated analysis of sequences of microscope images and machine-learning analysis to characterize cell morphology. As an aid in the quantitative understanding of these experiments, we have developed a computational framework for simulating the time dependence of sickling in populations of red blood cells which incorporates the current theoretical and empirical understanding of the physical chemistry of the sickling process. In order to apply these techniques to our experiments, we have theoretically determined the time course of deoxygenation by solving the diffusion equation for oxygen in our experimental geometry. With this combined description we are able to reproduce our experimentally observed kinetics of sickling, suggesting that our theoretical approach should be applicable to physiological deoxygenation scenarios.

Background. Sickle cell disease (SCD) is a devastating disorder initiated by polymerization of the deoxy-hemoglobin S (HbS) to form fibers that distort (sickle) erythrocytes. 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. Methods. Subjects aged ≥ 18 years with a confirmed diagnosis of SCD (HbSS), adequate organ function, baseline Hb ≥ 7 g/dL, and no transfusions or erythropoietin therapy in the prior 3 months were eligible. Concomitant stable hydroxyurea (HU) and/or L-Glutamine therapy was permitted on study. Subjects received either 3 or 4 ascending dose levels of mitapivat (5 mg BID, 20 mg BID, 50 mg BID, 100 mg BID) for 2 weeks' duration each, followed by a 12-15-day drug taper. The primary endpoint was safety and tolerability as assessed by frequency and severity of adverse events (AEs) and laboratory parameters. Secondary endpoints included changes in hematological parameters, 2,3-DPG and ATP levels, and markers of Hb S polymerization (p50 and t50). Results. Nine subjects have been enrolled as of July 2020. One patient discontinued treatment early and was lost to follow-up. Results pertain to the 8 subjects who completed study: mean age was 43.6 years (range 34-55 years), 5 were male, and 7 were stabilized on HU. The initial 6 subjects escalated to a maximum dose of 50 mg BID, and the subsequent 2 subjects escalated up to a 100 mg BID dose following a protocol amendment. All 8 subjects reported AEs; the most common treatment related AEs were hypertension (n=3, Grades 1-3), insomnia (n=3, Grade 2), and asymptomatic heart rate increase on vital signs (n=3, Grade 1). 5 serious AEs (SAEs) were reported in 4 subjects, with only 1 SAE possibly attributable to study drug - a vaso-occlusive crisis (VOC) during drug taper, after which the protocol was amended to increase the length of taper from 9 to 12 days for the 50 mg BID dose and 15 days for the 100 mg BID dose. No VOCs have occurred during core period or on the prolonged drug taper. The SAEs not attributable to study drug included a VOC at the end of study, VOC-related transaminitis (Grade 2), a non-VOC-related pain episode, and a pre-existing pulmonary embolism discovered shortly after study drug initiation. Table 1 details the PD and clinical laboratory measures. Mean 2,3-DPG levels decreased and ATP levels increased in a dose-dependent manner. ATP levels remained elevated immediately following drug taper in 5/8 subjects. Both ATP and 2,3-DPG levels returned to near baseline by 4 weeks after drug taper. Decreases in p50 and increases in t50 at various dose levels support an anti-sickling effect of mitapivat in a subset of subjects, though responses were highly variable. Mitapivat increased Hb levels in a dose-dependent manner with a concomitant decrease in hemolytic markers. Mean change in Hb from baseline for all 8 subjects at the 50 mg BID dose level was 1.2 g/dL (range -0.3-2.7 g/dL), followed by near return to mean Hb baseline after drug taper. Hemolytic markers similarly returned to near baseline after drug taper. 3/8 and 5/8 subjects achieved a Hb increase of ≥ 1 g/dL at the 20 and 50 mg BID dose levels, respectively. A small dose-dependent increase in mean corpuscular volume (MCV) was also observed, suggesting a potential alternative mechanism of mitapivat, acting through the ATP-dependent Gardos channel to increase red cell hydration, decrease hemolysis, and improve red cell survival in SCD. Conclusion. 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 completing 6-8 weeks of treatment. Additional data including PK will be presented. Disclosures Iyer: Novartis: Current equity holder in publicly-traded company; Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Mangus:Agios Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Kung:Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dang:Agios Pharmaceuticals Inc.: Current Employment, Current equity holder in publicly-traded company. Kosinski:Agios Pharmaceuticals Inc: Current Employment, Current equity holder in publicly-traded company. Hawkins:Infinity Pharmaceuticals: Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company; Jazz Pharmaceuticals: Current equity holder in publicly-traded company.

A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

Although it has been known for more than 60 years that the cause of sickle cell disease is polymerization of a hemoglobin mutant, hydroxyurea is the only drug approved for treatment by the US Food and Drug Administration. This drug, however, is only partially successful, and the discovery of additional drugs that inhibit fiber formation has been hampered by the lack of a sensitive and quantitative cellular assay. Here, we describe such a method in a 96-well plate format that is based on laser-induced polymerization in sickle trait cells and robust, automated image analysis to detect the precise time at which fibers distort ("sickle") the cells. With this kinetic method, we show that small increases in cell volume to reduce the hemoglobin concentration can result in therapeutic increases in the delay time prior to fiber formation. We also show that, of the two drugs (AES103 and GBT440) in clinical trials that inhibit polymerization by increasing oxygen affinity, one of them (GBT440) also inhibits sickling in the absence of oxygen by two additional mechanisms.

Deamidation of asparagine and glutamine is the most common non-enzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers which have an altered structure, but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.

We report that the waiting time delay in 2D IR pulse sequences can be used to suppress signals from structurally disordered regions of amyloid fibrils. At a waiting time delay of 1.0 ps, the random coil vibrational modes of amylin fibrils are no longer detectable, leaving only the sharp excitonic vibrational features of the fibril β-sheets. Isotope labeling with (13)C(18)O reveals that structurally disordered residues decay faster than residues protected from solvent. Since structural disorder is usually accompanied by hydration, we conclude that the shorter lifetimes of random-coil residues is due to solvent exposure. These results indicate that 2D IR pulse sequences can utilize the waiting time to better resolve solvent-protected regions of peptides and that local mode lifetimes should be included in simulations of 2D IR spectra.

Transition dipoles are an underutilized quantity for probing molecular structures. The transition dipole strengths in an extended system like a protein are modulated by the couplings and thus probe the structures. Here we measure the absolute transition dipole strengths of human and rat amylin in their solution, aggregated, membrane, and micelleular bound forms, using a combination of 1D and 2D infrared spectroscopy. We find that the vibrational modes of amyloid fibers made of human amylin can extend across as many as 12 amino acids, reflecting very ordered β-sheets in the most carefully prepared samples. Rat amylin has FTIR spectra that are nearly identical in solution, micelles, and membranes. We show that the transition dipoles of rat amylin are much larger when bound to micelles and membranes than when in solution, consistent with rat amylin adopting an α-helical structure. We interpret the transition dipole strengths as experimental measurements of the inverse participation ratio often calculated in theoretical studies. The structure of aggregating and membrane-bound proteins can be difficult to identify with existing techniques, especially during kinetics. These results demonstrate how absolute transition dipoles measured via our 1D/ 2D spectroscopy method can provide important structural information.

The combination of infrared spectroscopy and isotope labeling is a powerful toolset for providing residue-specific information about the structure and aggregation kinetics of amyloid peptides. In this chapter, we review a simple mathematical formalism to guide the interpretation of 1D- and 2DIR spectra of amyloid fibers. This formalism enables the design of isotope labeling schemes to extract precise structural features from IR spectra. We present 2DIR experiments on hIAPP and Aβ in which some of these strategies have been employed.