Functional and multi-omics signatures of mitapivat efficacy upon activation of pyruvate kinase in red blood cells from patients with sickle cell disease.

Mitapivat, a pyruvate kinase activator, shows great potential as a sickle cell disease (SCD)-modifying therapy. The safety and efficacy of mitapivat as a long-term maintenance therapy are currently being evaluated in two open-label studies. Here we applied a comprehensive multi-omics approach to investigate the impact of activating pyruvate kinase on red blood cells (RBC) from 15 SCD patients. HbSS patients were enrolled in one of the open-label, extended studies (NCT04610866). Leukodepleted RBC obtained from fresh whole blood at baseline, prior to drug initiation, and at longitudinal timepoints over the course of the study were processed for multi-omics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBC. Mitapivat decreased 2,3-diphosphoglycerate levels, increased adenosine triphosphate levels, and improved hematologic and sickling parameters in patients with SCD. Agreement between omics measurements and clinical measurements confirmed the specificity of mitapivat on targeting late glycolysis, with glycolytic metabolites ranking as the top correlates to parameters of hemoglobin S oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of treatment, with minimal changes in reticulocyte counts. In the first 6 months of treatment there were also transient elevations of lysophosphatidylcholines and oxylipins with depletion of free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBC identified benefits for glycolysis, as well as activation of the Lands cycle.

In vivo measurement of RBC survival in patients with sickle cell disease before or after hematopoietic stem cell transplantation.

ABSTRACT: Stable, mixed-donor-recipient chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with sickle cell disease (SCD) is sufficient for phenotypic disease reversal, and results from differences in donor/recipient-red blood cell (RBC) survival. Understanding variability and predictors of RBC survival among patients with SCD before and after HSCT is critical for gene therapy research which seeks to generate sufficient corrected hemoglobin to reduce polymerization thereby overcoming the red cell pathology of SCD. This study used biotin labeling of RBCs to determine the lifespan of RBCs in patients with SCD compared with patients who have successfully undergone curative HSCT, participants with sickle cell trait (HbAS), and healthy (HbAA) donors. Twenty participants were included in the analysis (SCD pre-HSCT: N = 6, SCD post-HSCT: N = 5, HbAS: N = 6, and HbAA: N = 3). The average RBC lifespan was significantly shorter for participants with SCD pre-HSCT (64.1 days; range, 35-91) compared with those with SCD post-HSCT (113.4 days; range, 105-119), HbAS (126.0 days; range, 119-147), and HbAA (123.7 days; range, 91-147) (P<.001). RBC lifespan correlated with various hematologic parameters and strongly correlated with the average final fraction of sickled RBCs after deoxygenation (P<.001). No adverse events were attributable to the use of biotin and related procedures. Biotin labeling of RBCs is a safe and feasible methodology to evaluate RBC survival in patients with SCD before and after HSCT. Understanding differences in RBC survival may ultimately guide gene therapy protocols to determine hemoglobin composition required to reverse the SCD phenotype as it relates directly to RBC survival. This trial was registered at www.clinicaltrials.gov as #NCT04476277.

Recent developments in the use of pyruvate kinase activators as a new approach for treating sickle cell disease.

ABSTRACT: Pyruvate kinase (PK) is a key enzyme in glycolysis, the sole source of adenosine triphosphate, which is essential for all energy-dependent activities of red blood cells. Activating PK shows great potential for treating a broad range of hemolytic anemias beyond PK deficiency, because they also enhance activity of wild-type PK. Motivated by observations of sickle-cell complications in sickle-trait individuals with concomitant PK deficiency, activating endogenous PK offers a novel and promising approach for treating patients with sickle-cell disease.

Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease.

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 for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the 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 antisickling 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 antisickling 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 potential 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.

A phase 1 dose escalation study of the pyruvate kinase activator mitapivat (AG-348) in sickle cell disease.

Polymerization of deoxygenated hemoglobin S underlies the pathophysiology of sickle cell disease (SCD). In activating red blood cell pyruvate kinase and glycolysis, mitapivat (AG-348) increases adenosine triphosphate (ATP) levels and decreases the 2,3-diphosphoglycerate (2,3-DPG) concentration, an upstream precursor in glycolysis. Both changes have therapeutic potential for patients with SCD. Here, we evaluated the safety and tolerability of multiple ascending doses of mitapivat in adults with SCD with no recent blood transfusions or changes in hydroxyurea or l-glutamine therapy. Seventeen subjects were enrolled; 1 subject was withdrawn shortly after starting the study. Sixteen subjects completed 3 ascending dose levels of mitapivat (5, 20, and 50 mg, twice daily [BID]) for 2 weeks each; following a protocol amendment, the dose was escalated to 100 mg BID in 9 subjects. Mitapivat was well tolerated at all dose levels, with the most common treatment-emergent adverse events (AEs) being insomnia, headache, and hypertension. Six serious AEs (SAEs) included 4 vaso-occlusive crises (VOCs), non-VOC-related shoulder pain, and a preexisting pulmonary embolism. Two VOCs occurred during drug taper and were possibly drug related; no other SAEs were drug related. Mean hemoglobin increase at the 50 mg BID dose level was 1.2 g/dL, with 9 of 16 (56.3%) patients achieving a hemoglobin response of a ≥1 g/dL increase compared with baseline. Mean reductions in hemolytic markers and dose-dependent decreases in 2,3-DPG and increases in ATP were also observed. This study provides proof of concept that mitapivat has disease-modifying potential in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

Mitapivat increases ATP and decreases oxidative stress and erythrocyte mitochondria retention in a SCD mouse model.

Polymerization of deoxygenated sickle hemoglobin (HbS) leads to erythrocyte sickling. Enhancing activity of the erythrocyte glycolytic pathway has anti-sickling potential as this reduces 2,3-diphosphoglycerate (2,3-DPG) and increases ATP, factors that decrease HbS polymerization and improve erythrocyte membrane integrity. These factors can be modulated by mitapivat, which activates erythrocyte pyruvate kinase (PKR) and improves sickling kinetics in SCD patients. We investigated mechanisms by which mitapivat may impact SCD by examining its effects in the Townes SCD mouse model. Control (HbAA) and sickle (HbSS) mice were treated with mitapivat or vehicle. Surprisingly, HbSS had higher PKR protein, higher ATP, and lower 2,3-DPG levels, compared to HbAA mice, in contrast with humans with SCD, in whom 2,3-DPG is elevated compared to healthy subjects. Despite our inability to investigate 2,3-DPG-mediated sickling and hemoglobin effects, mitapivat yielded potential benefits in HbSS mice. Mitapivat further increased ATP without significantly changing 2,3-DPG or hemoglobin levels, and decreased levels of leukocytosis, erythrocyte oxidative stress, and the percentage of erythrocytes that retained mitochondria in HbSS mice. These data suggest that, even though Townes HbSS mice have increased PKR activity, further activation of PKR with mitapivat yields potentially beneficial effects that are independent of changes in sickling or hemoglobin levels.

Distinguishing between protein dynamics and dye photophysics in single-molecule FRET experiments.

Förster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information. Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e., protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification. The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise. Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity.

Evidence for a partially structured state of the amylin monomer.

Islet amyloid polypeptide (amylin) is the main component in amyloid deposits formed in type II diabetes. We used triplet quenching to probe the dynamics of contact formation between the N-terminal disulfide loop and a C-terminal tryptophan in monomeric amylins from human and rat. Quenching rates measured in the absence of denaturant are four times larger than those in 6 M guanidinium chloride, indicating a decrease in the average end-to-end distance (collapse) at low denaturant concentrations. We were surprised to find an even greater (sevenfold) increase in quenching rates on removal of denaturant for a hydrophilic control peptide containing the disulfide loop compared to the same peptide without the loop (twofold change). These results suggest that collapse is driven by backbone-backbone and backbone-side chain interactions involving the disulfide loop portion of the chain rather than by the formation of side-chain hydrophobic contacts. Molecular dynamics simulations of the control peptide show that the collapse results from hydrogen-bonding interactions between the central residues of the chain and the disulfide loop. The quenching experiments also indicate that the monomer of the human, amyloidogenic form of amylin is more compact than the rat form, which does not form amyloid. We discuss these newly observed differences between human and rat amylin in solution and their possible relation to aggregation and to the physiological function of amylin binding to the calcitonin receptor.

Sedimentation studies on human amylin fail to detect low-molecular-weight oligomers.

Sedimentation velocity experiments show that only monomers coexist with amyloid fibrils of human islet amyloid-polypeptide. No oligomers containing <100 monomers could be detected, suggesting that the putative toxic oligomers are much larger than those found for the Alzheimer's peptide, Abeta(1-42).

Effect of flexibility and cis residues in single-molecule FRET studies of polyproline.

Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Förster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to the flexibility of the polypeptide. Here, we investigate this issue further, using a combination of single-molecule fluorescence intensity and lifetime measurements, NMR, theory, and molecular dynamics simulations of polyproline-20 that include the dyes and their linkers to the polypeptide. NMR shows that in water approximately 30% of the molecules contain internal cis prolines, whereas none are detectable in trifluoroethanol. Simulations suggest that the all-trans form of polyproline is relatively stiff, with persistence lengths of 9-13 nm using different established force fields, and that the kinks arising from internal cis prolines are primarily responsible for the higher mean FRET efficiency in water. We show that the observed efficiency histograms and distributions of donor fluorescence lifetimes are explained by the presence of multiple species with efficiencies consistent with the simulations and populations determined by NMR. In calculating FRET efficiencies from the simulation, we find that the fluctuations of the chromophores, attached to long flexible linkers, also play an important role. A similar simulation approach suggests that the flexibility of the chromophore linkers is largely responsible for the previously unexplained high value of R(0) required to fit the data in the classic study of Stryer and Haugland.

Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations.

To obtain quantitative information on the size and dynamics of unfolded proteins we combined single-molecule lifetime and intensity FRET measurements with molecular simulations. We compared the unfolded states of the 64-residue, alpha/beta protein L and the 66-residue, all-beta cold-shock protein CspTm. The average radius of gyration (Rg) calculated from FRET data on freely diffusing molecules was identical for the two unfolded proteins at guanidinium chloride concentrations >3 M, and the FRET-derived Rg of protein L agreed well with the Rg previously measured by equilibrium small-angle x-ray scattering. As the denaturant concentration was lowered, the mean FRET efficiency of the unfolded subpopulation increased, signaling collapse of the polypeptide chain, with protein L being slightly more compact than CspTm. A decrease in Rg with decreasing denaturant was also observed in all-atom molecular dynamics calculations in explicit water/urea solvent, and Langevin simulations of a simplified representation of the polypeptide suggest that collapse can result from either increased interresidue attraction or decreased excluded volume. In contrast to both the FRET and simulation results, previous time-resolved small-angle x-ray scattering experiments showed no collapse for protein L. Analysis of the donor fluorescence decay of the unfolded subpopulation of both proteins gives information about the end-to-end chain distribution and suggests that chain dynamics is slow compared with the donor life-time of approximately 2 ns, whereas the bin-size independence of the small excess width above the shot noise for the FRET efficiency distributions may result from incomplete conformational averaging on even the 1-ms time scale.

Folding and aggregation kinetics of a beta-hairpin.

We have investigated the solution structure, equilibrium properties, and folding kinetics of a 17-residue beta-hairpin-forming peptide derived from the protein ubiquitin. NMR experiments show that at 4 degrees C the peptide has a highly populated beta-hairpin conformation. At protein concentrations higher than 0.35 mM, the peptide aggregates. Sedimentation equilibrium measurements show that the aggregate is a trimer, while NMR indicates that the beta-hairpin conformation is maintained in the trimer. The relaxation kinetics in nanosecond laser temperature-jump experiments reveal a concentration-independent microsecond phase, corresponding to beta-hairpin unfolding-refolding, and a concentration-dependent millisecond phase due to oligomerization. Kinetic modeling of the relaxation rates and amplitudes yields the folding and unfolding rates for the monomeric beta-hairpin, as well as assembly and disassembly rates for trimer formation consistent with the equilibrium constant determined by sedimentation equilibrium. When the net charge on the peptides and ionic strength were taken into account, the rate of trimer assembly approaches the Debye-Smoluchowski diffusion limit. At 300 K, the rate of formation of the monomeric hairpin is (17 micros)(-1), compared to rates of (0.8 micros)(-1) to (52 micros)(-1) found for other peptides. After using Kramers theory to correct for the temperature dependence of the pre-exponential factor, the activation energy for hairpin formation is near zero, indicating that the barrier to folding is purely entropic. Comparisons with previously measured rates for a series of hairpins are made to distinguish between zipper and hydrophobic collapse mechanisms. Overall, the experimental data are most consistent with the zipper mechanism in which structure formation is initiated at the turn, the mechanism predicted by the Ising-like statistical mechanical model that was developed to explain the equilibrium and kinetic data for the beta-hairpin from protein GB1. In contrast, the majority of simulation studies favor a hydrophobic collapse mechanism. However, with few exceptions, there is little or no quantitative comparison of the simulation results with experimental data.

Effects of denaturants on the dynamics of loop formation in polypeptides.

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)j-trp (j=1-9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4-0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from approximately 0.6 nm to approximately 0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50-400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.

Determination of ultrafast protein folding rates from loop formation dynamics.

Quenching of the triplet state of tryptophan by contact with cysteine can be used to measure the kinetics of loop formation in unfolded proteins. Here we show that cysteine quenching dynamics also provide a novel method for measuring folding rates when the exchange between folded and unfolded states is faster than the unquenched triplet lifetime (approximately 100 micros). We use this technique to investigate folding/unfolding kinetics of the 35 residue headpiece subdomain of the protein villin, which contains a single tryptophan residue and was engineered to contain a cysteine residue at the N terminus. At intermediate concentrations of denaturant the time-course of the triplet decay consists of two relaxations, the rates and amplitudes of which reveal the fast kinetics for folding and unfolding of this protein. The folding rates extracted using a simple kinetic model are close to those reported previously from laser-induced temperature-jump experiments that employ the change in tryptophan fluorescence as a probe. However, the results differ significantly from those reported from dynamic NMR line shape analysis on a variant with methionine at the N terminus, an issue that remains to be resolved. The analysis of the triplet quenching kinetics also shows that the quenching rates in the unfolded state increase with decreasing denaturant concentration, indicating a compaction of the unfolded protein.

Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence.

To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R(0), the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R(0), they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances.

Kinetics of intramolecular contact formation in a denatured protein.

Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein ( approximately 10(4)s(-1)). Experiments on the unfolded protein without the added C-terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule Förster resonance energy transfer (FRET) experiments.

Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy.

Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer (FRET) has been measured on single, freely diffusing molecules. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.

Measuring dynamic flexibility of the coil state of a helix-forming peptide.

To investigate the dynamic flexibility of the coil state of a helix-forming peptide the end-to-end contact rate was determined. Nanosecond optical excitation of tryptophan at one end of a 22 residue, alanine peptide populates a long-lived triplet state which is quenched upon close contact with a cyclic disulfide attached to the opposite end. Analysis of the decay of the triplet population using a two-state model for helix formation yields the diffusion-limited end-to-end contact rate of the coil state of the peptide as well as the helix-->coil and coil-->helix rates. The helix-coil rates are very similar to those previously measured in laser temperature-jump experiments. The end-to-end contact rate of 1.1 x 10(7) s(-1) in the coil state is tenfold faster than the rate for a disordered peptide with threonine substituted for alanine and, somewhat surprisingly, is about twice the rate for a disordered glycine-containing peptide. These differences are discussed in terms of the theory of Szabo, Schulten and Schulten. The rates should provide important new benchmarks for testing the accuracy of atomistic molecular dynamics simulations.