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.

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 (PK) activator, shows great potential as a sickle cell disease (SCD)- modifying therapy. Safety and efficacy of mitapivat as a long-term maintenance therapy is currently being evaluated in two open-label studies. Here we apply a comprehensive multi-omics approach to investigate the impact of activating PK on red blood cells (RBCs) from 15 SCD patients. HbSS patients were enrolled in one of the open label, extended studies (NCT04610866). Leuko-depleted RBCs obtained from fresh whole blood at baseline (visit 1, V1), prior to drug initiation and longitudinal time points over the course of the study were processed for multiomics through a stepwise extraction of metabolites, lipids and proteins. Mitapivat therapy had significant effects on the metabolome, lipidome and proteome of SCD RBCs. Mitapivat decreased 2,3-diphosphoglycerate (DPG) levels, increased adenosine triphosphate (ATP) 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 (HbS) oxygen affinity (p50) and sickling kinetics (t50) during treatment. Mitapivat markedly reduced levels of proteins of mitochondrial origin within 2 weeks of initiation of drug treatment, with minimal changes in the reticulocyte counts. The first six months of treatment also witnessed transient elevation of lysophosphatidylcholines and oxylipins with depletion in free fatty acids, suggestive of an effect on membrane lipid remodeling. Multi-omics analysis of RBCs identified benefits for glycolysis, as well as activation of the Lands cycle.

Estimating computational limits on theoretical descriptions of biological cells.

There has been much success recently in theoretically simulating parts of complex biological systems on the molecular level, with the goal of first-principles modeling of whole cells. However, there is the question of whether such simulations can be performed because of the enormous complexity of cells. We establish approximate equations to estimate computation times required to simulate highly simplified models of cells by either molecular dynamics calculations or by solving molecular kinetic equations. Our equations place limits on the complexity of cells that can be theoretically understood with these two methods and provide a first step in developing what can be considered biological uncertainty relations for molecular models of cells. While a molecular kinetics description of the genetically simplest bacterial cell may indeed soon be possible, neither theoretical description for a multicellular system, such as the human brain, will be possible for many decades and may never be possible even with quantum computing.

Treatment of sickle cell disease by increasing oxygen affinity of hemoglobin.

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.

Psychiatric comorbidity in the Baltimore ECA follow-up study: the matrix approach.

BACKGROUND: Psychiatric comorbidity is defined as the joint occurrence of two or more mental or substance use disorders. Widespread psychiatric comorbidity has been reported in treatment and population-based studies. The aim of this study was to measure the extent and impact of psychiatric comorbidity in a cohort of the Baltimore Epidemiologic Catchment Area study. METHODS: We examined the comorbidity burden of 16 mental disorders in a cohort of 847 participants using both established and novel analytical approaches The Comorbidity to Diagnosis Inflation Ratio (CDIR), is a statistical instrument that quantifies impact of pairwise comorbid associations, both on the whole sample, as well as on each specific disorder. RESULTS: Most anxiety disorders had substantial co-occurrence with each other, as well as with Major Depressive Disorder (MDD). In addition, mood disorders had a high degree of comorbidity with Alcohol Dependence (AD). The CDIR for the whole sample was 1.32, indicating a ratio of 132 comorbidities per 100 diagnoses. The conditions with high sample prevalence were relatively less comorbid than the low prevalence conditions. Obsessive Compulsive Disorder had a comorbidity burden that was 89% greater than the overall sample. CONCLUSION: Anxiety disorders are highly interrelated, as well as highly comorbid with depression. The comorbidity phenomenon is linked to the differential prevalence of the analyzed conditions. Comorbidity frequency (most prevalent comorbid condition) appears mutually exclusive to comorbidity burden (most widely interrelated condition). While AD and MDD were the most frequently diagnosed disorders; low prevalence conditions as OCD and GAD were the most widely interrelated.

Physics of virus transmission by speaking droplets.

To make the physics of person-to-person virus transmission from emitted droplets of oral fluid while speaking easily understood, we present simple and transparent algebraic equations that capture the essential physics of the problem. Calculations with these equations provide a straightforward way of determining whether emitted droplets remain airborne or rapidly fall to the ground, after accounting for the decrease in droplet size from water evaporation. At a relative humidity of 50%, for example, droplets with initial radii larger than about 50 µm rapidly fall to the ground, while smaller, potentially virus-containing droplets shrink in size from water evaporation and remain airborne for many minutes. Estimates of airborne virion emission rates while speaking strongly support the proposal that mouth coverings can help contain the COVID-19 pandemic.

Allosteric control of hemoglobin S fiber formation by oxygen and its relation to the pathophysiology of sickle cell disease.

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.

Association of common infections with cognitive performance in the Baltimore Epidemiologic Catchment Area study follow-up.

INTRODUCTION: Growing evidence suggests that some common infections are causally associated with cognitive impairment; however, less is known about the burden of multiple infections. METHODS: We investigated the cross-sectional association of positive antibody tests for herpes simplex virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), and Toxoplasma gondii (TOX) with Mini-Mental State Examination (MMSE) and delayed verbal recall performance in 575 adults aged 41-97 from the Baltimore Epidemiologic Catchment Area Study. RESULTS: In multivariable-adjusted zero-inflated Poisson (ZIP) regression models, positive antibody tests for CMV (p = .011) and herpes simplex virus (p = .018) were individually associated with poorer MMSE performance (p = .011). A greater number of positive antibody tests among the five tested was associated with worse MMSE performance (p = .001). DISCUSSION: CMV, herpes simplex virus, and the global burden of multiple common infections were independently associated with poorer cognitive performance. Additional research that investigates whether the global burden of infection predicts cognitive decline and Alzheimer's disease biomarker changes is needed to confirm these findings.

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.

From population to neuron: exploring common mediators for metabolic problems and mental illnesses.

Major mental illnesses such as schizophrenia (SZ) and bipolar disorder (BP) frequently accompany metabolic conditions, but their relationship is still unclear, in particular at the mechanistic level. We implemented an approach of "from population to neuron", combining population-based epidemiological analysis with neurobiological experiments using cell and animal models based on a hypothesis built from the epidemiological study. We characterized high-quality population data, olfactory neuronal cells biopsied from patients with SZ or BP, and healthy subjects, as well as mice genetically modified for insulin signaling. We accessed the Danish Registry and observed (1) a higher incidence of diabetes in people with SZ or BP and (2) higher incidence of major mental illnesses in people with diabetes in the same large cohort. These epidemiological data suggest the existence of common pathophysiological mediators in both diabetes and major mental illnesses. We hypothesized that molecules associated with insulin resistance might be such common mediators, and then validated the hypothesis by using two independent sets of olfactory neuronal cells biopsied from patients and healthy controls. In the first set, we confirmed an enrichment of insulin signaling-associated molecules among the genes that were significantly different between SZ patients and controls in unbiased expression profiling data. In the second set, olfactory neuronal cells from SZ and BP patients who were not pre-diabetic or diabetic showed reduced IRS2 tyrosine phosphorylation upon insulin stimulation, indicative of insulin resistance. These cells also displayed an upregulation of IRS1 protein phosphorylation at serine-312 at baseline (without insulin stimulation), further supporting the concept of insulin resistance in olfactory neuronal cells from SZ patients. Finally, Irs2 knockout mice showed an aberrant response to amphetamine, which is also observed in some patients with major mental illnesses. The bi-directional relationships between major mental illnesses and diabetes suggest that there may be common pathophysiological mediators associated with insulin resistance underlying these mental and physical conditions.

A retrospective on statistical mechanical models for hemoglobin allostery.

Understanding allosteric interactions in proteins has become one of the major research areas in protein science. The original aim of the famous theoretical model of Monod, Wyman, and Changeux (MWC) was to explain the regulation of enzymatic activity in biochemical pathways. However, its first successful quantitative application was to explain cooperative oxygen binding by hemoglobin, often called the "hydrogen molecule of biology." The combination of its original application and the enormous amount of research on hemoglobin has made it the paradigm for studies of allostery, especially for multi-subunit proteins, and for the development of statistical mechanical models to describe how structure determines function. This article is a historical account of the development of statistical mechanical models for hemoglobin to explain both the cooperative binding of oxygen (called homotropic effects by MWC) and how oxygen binding is affected by ligands that bind distant from the heme oxygen binding site (called heterotropic allosteric effects by MWC). This account makes clear the many remaining challenges for describing the relationship of structure to function for hemoglobin in terms of a satisfactory statistical mechanical model.

The Relationship Between Latinxs' Acculturative Experiences and Mental and Behavioral Disorder in the National Latino and Asian American Study.

Mental and behavioral disorders are among the leading contributors to disability among US-residing Latinxs. When treated as a homogeneous group, important disparities in the prevalence of such disorders among Latinx subgroups (e.g., by ethnic heritage) are obscured. However, Latinxs may also be characterized by their acculturative experiences while living in the USA, such as discrimination, neighborhood context and family conflict. Latent Profile Analysis with distal outcomes was used to estimate differences in psychiatric disorder prevalence across acculturative subgroups. Data from 2,541 Latinx participants (age 18 +) in the National Latino and Asian American Study (NLAAS) were used to estimate differences in the proportion of three categories of DSM-IV disorder: depressive, anxiety and substance use by four latent subgroups of Latinxs based on their acculturative experiences. Latinxs reporting more positive acculturative experiences had the lowest prevalence of all three disorders (14.8%, 13.6% and 7.1%, respectively). Those whose lives were characterized by high levels of family conflict and discrimination combined with low levels of social cohesion and neighborhood safety had the highest disorder prevalence (34.0%, 26.6% and 22.5%; all p < 0.01 compared to positive experiences subgroup). Latinxs with moderate levels of discrimination and conflict, along with those with high conflict and cohesion, were better off as compared to those with high negative experiences and low cohesion. These latent subgroups of Latinxs according to their acculturative experiences hold important implications for identifying high-risk groups for developing a psychiatric disorder. Findings also point to the protective role of family and neighborhood cohesion when facing high levels of adversity, which may inform prevention and intervention efforts.

MWC allosteric model explains unusual hemoglobin-oxygen binding curves from sickle cell drug binding.

An oxygen-affinity-modifying drug, voxelotor, has very recently been approved by the FDA for treatment of sickle cell disease. The proposed mechanism of action is by preferential binding of the drug to the R quaternary conformation, which cannot copolymerize with the T conformation to form sickle fibers. Here, we report widely different oxygen dissociation and oxygen association curves for normal blood in the presence of voxelotor and interpret the results in terms of the allosteric model of Monod, Wyman, and Changeux with the addition of drug binding. The model does remarkably well in quantitatively explaining a complex data set with just the addition of drug binding and dissociation rates for the R and T conformations. Whereas slow dissociation of the drug from R results in time-independent dissociation curves, the changing association curves result from slow dissociation of the drug from T, as well as extremely slow binding of the drug to T. By calculating true equilibrium curves from the model parameters, we show that there would be a smaller decrease in oxygen delivery from the left shift in the dissociation curve caused by drug binding if drug binding and dissociation for both R and T were rapid. Our application of the Monod, Wyman, and Changeux model demonstrates once more its enormous power in explaining many different kinds of experimental results for hemoglobin. It should also be helpful in analyzing oxygen binding and in vivo delivery in future investigations of oxygen-affinity-modifying drugs for sickle cell disease.

Impact of Conformational Substates and Energy Landscapes on Understanding Hemoglobin Kinetics and Function.

Hans Frauenfelder's discovery of conformational substates in studies of myoglobin carbon monoxide geminate rebinding kinetics at cryogenic temperatures (Austin RH, Beeson KW, Eisenstein L, Frauenfelder H, & Gunsalus IC (1975) Dynamics of Ligand Binding to Myoglobin. Biochemistry 14(24):5355-5373) followed by his introduction of energy landscape theory with Peter Wolynes (Frauenfelder H, Sligar SG, & Wolynes PG (1991) The Energy Landscapes and Motions of Proteins. Science 254(5038):1598-1603) marked the beginning of a new era in the physics and physical chemistry of proteins. Their work played a major role in demonstrating the power and importance of dynamics and of Kramers reaction rate theory for understanding protein function. The biggest impact of energy landscape theory has been in the protein folding field, which is well-known and has been documented in numerous articles and reviews, including a recent one of my own (Eaton WA (2021) Modern Kinetics and Mechanism of Protein Folding: a Retrospective. J. Phys. Chem. B. 125(14):3452-3467). Here I will describe the much less well-known impact of their modern view of proteins on both experimental and theoretical studies of hemoglobin kinetics and function. I will first describe how Frauenfelder's experiments motivated and influenced my own research on myoglobin, which were key ingredients to my work on understanding hemoglobin.

Genetic propensity for risky behavior and depression and risk of lifetime suicide attempt among urban African Americans in adolescence and young adulthood.

Suicide attempts (SA) among African Americans have increased at a greater rate than any other racial/ethnic group. Research in European ancestry populations has indicated that SA are genetically influenced; however, less is known about the genetic contributors that underpin SA among African Americans. We examined whether genetic propensity for depression and risky behaviors (assessed via polygenic risk scores; PRS) independently and jointly are associated with SA among urban, African Americans and whether sex differences exist in these relations. Participants (N = 1,157, 45.0% male) were originally recruited as part of two first grade universal school-based prevention trials. Participants reported in adolescence and young adulthood on whether they ever attempted suicide in their life. Depression and risky behaviors PRS were created based on large-scale genome-wide association studies conducted by Howard et al. (2019) and Karlson Línner et al. (2019), respectively. There was a significant interaction between the risky behavior PRS and depression PRS such that the combination of high risky behavior polygenic risk and low/moderate polygenic risk for depression was associated with greater risk for lifetime SA among the whole sample and African American males specifically. In addition, the risky behavior PRS was significantly positively associated with lifetime SA among African American males. These findings provide preliminary evidence regarding the importance of examining risky behavior and depression polygenic risk in relation to SA among African Americans, though replication of our findings in other African American samples is needed.

Hemoglobin S polymerization and sickle cell disease: A retrospective on the occasion of the 70th anniversary of Pauling's Science paper.

70 years ago, Linus Pauling, the legendary genius of 20th century chemistry, published his famous work on the molecular cause of sickle cell disease, a paper that gave birth to what is now called molecular medicine. In this paper, Pauling left important questions unanswered that have motivated an enormous amount of scientific and clinical research since then. This retrospective discusses the basic science studies that have answered those questions directly related to the kinetics and thermodynamics of hemoglobin S polymerization.

The serotonin transporter gene polymorphism (SLC6A4) and risk for psychiatric morbidity and comorbidity in the Baltimore ECA follow-up study.

BACKGROUND: The human serotonin transporter (SERT) gene polymorphism (5HTTLPR) has been associated with multiple psychiatric disorders, including major depression, anxiety disorders, and substance use disorders. This study investigated the association between 5HTTLPR and psychiatric morbidity and comorbidity in a psychiatrist-examined population sample. METHODS: 628 participants, mean age 48.3 years old, were assessed by psychiatrists using the Schedules for Clinical Assessment in Neuropsychiatry. Associations between 5HTTLPR and the prevalence, comorbidity, and time-to-diagnoses for 16 psychiatric conditions were evaluated, using several analytical approaches. RESULTS: The SERT S allele was significantly associated with an increased lifetime prevalence of panic disorder. There was a "protective" association between SERT gene S allele carrier status and the risk of obsessive-compulsive disorder (OCD) in time-to-event analysis. Carriers of the S allele had a significant increased risk of two specific comorbid disorder pairs: major depressive disorder (MDD) and social phobia, and MDD and agoraphobia. Overall, there was no increased risk of receiving an initial or an additional diagnosis for a mental disorder in the SERT S allele carriers CONCLUSIONS: The findings suggest that the S allele carrier status is associated with an increased prevalence of panic disorder in a community sample. There was an increased risk for comorbidity in a more homogeneous subgroup of cases with MDD and social phobia, as well as or agoraphobia. Our findings suggest a specific effect of the SERT promoter gene polymorphism on a subgroup of individuals identifiable by their comorbidity.

Kinetic assay shows that increasing red cell volume could be a treatment for sickle cell disease.

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.