Hemoglobin Variants as Targets for Stabilizing Drugs

1. Introduction

Hemoglobin (Hb) is a crucial protein for oxygen transport in mammals, with a complex structure and function. The cooperative binding of oxygen to Hb involves an equilibrium between two quaternary structures with different oxygen affinities [1,2]. This process is influenced by various factors, including pH (the Bohr effect), organic phosphates, and other allosteric effectors [3,4]. The two-state model proposed by Monod, Wyman, and Changeux has been widely used to explain Hb cooperativity, although some discrepancies have been observed [2].

Hb exhibits complex allosteric regulation, crucial for its oxygen-binding function. The interplay between tense and relaxed states governs Hb's behavior [5,6]. Allosteric effectors, both endogenous and synthetic, modulate Hb's oxygen affinity by binding to specific sites, altering its structure and function [7,8]. This allosteric control involves both homotropic and heterotropic effects, where ligand binding at one site influences binding at other sites [9]. Protein engineering strategies have explored mutations to optimize Hb's functional properties, revealing additive effects of interfacial and distal-heme pocket mutations on structure and function [10]. The evolution of Hb has resulted in diverse variants with altered oxygen-binding properties, while maintaining fundamental structural features [11]. Un[1,2derstanding these mechanisms is crucial for developing Hb-based oxygen carriers and treating blood-related disorders [12]. Hemoglobin oxygen affinity is influenced by several physiological ligands (Figure 1): (1) pH aka Bohr effect - a decrease in pH reduces hemoglobin’s oxygen affinity, promoting oxygen release in tissues [3]; (2) CO₂ levels - increased CO₂ levels form carbaminohemoglobin, stabilizing the T state and reducing oxygen affinity [13]; (3) NO, which can bind to Hb's heme groups, forming nitrosylhemoglobin, or to cysteine residues, creating S-nitrosohemoglobin (SNO-Hb) [14,15] and (4) 2,3-Bisphosphoglycerate (2,3-BPG) - this metabolite binds to deoxygenated hemoglobin, stabilizing the T state and promoting oxygen release in tissues. 2,3-Bisphosphoglycerate (2,3-BPG) is a crucial metabolite that binds to hemoglobin, regulating oxygen affinity and delivery to tissues. It binds more strongly to deoxyhemoglobin than oxyhemoglobin, with one binding site per tetramer [16] [17,18]. 2,3-BPG levels increase during hypoxia, enhancing oxygen delivery [19]. Its concentration is regulated by complex interactions between glycolysis, the 2,3-BPG shunt, and the pentose phosphate pathway [20]. Feedback inhibition of hexokinase and phosphofructokinase by 2,3-BPG, along with product inhibition of 2,3-BPG synthase, control its steady-state concentration. H+ and oxygen effectively regulate 2,3-BPG levels, primarily through hexokinase and phosphofructokinase. The 2,3-BPG shunt also plays a role in stabilizing ATP levels.

2. Hemoglobin Function and Clinical Variants

The α and β globin genes are located on different chromosomes, with α globin genes on chromosome 16 and β globin genes on chromosome 11. The arrangement and expression of these genes are tightly regulated to ensure proper hemoglobin formation and function. Two main natural variants exist: fetal hemoglobin (HbF, α2γ2) and adult hemoglobin (HbA, α2β2) [22]. HbF has higher oxygen affinity than HbA, facilitating oxygen transfer from mother to fetus [23]. During development, HbF levels decrease from 70% at birth to <1% in adults, while HbA becomes predominant [24,25]. HbA2 (α2δ2) comprises 1.5-3.5% of adult hemoglobin [26]. Genetic factors, including mutations and polymorphisms, can influence HbF production in adulthood [27]. Structural hemoglobinopathies and thalassemias result from mutations affecting globin genes, leading to abnormal or decreased hemoglobin production [28]. These variants can interfere with HbA1c measurements, impacting diabetes monitoring [29].

2.1. Diversity of Genetic Variants of Hemoglobin

Ca. 10 years ago, over 1000 naturally occurring hemoglobin variants have been identified; these variants show various effect hemoglobin structure and biochemical properties with varying physiological effects [30]. Since then, several new variants have been found and their sequences can be retrieved from UniProt database. UniProt entries for human hemoglobin α and β subunits are

https://www.uniprot.org/uniprotkb/P69905/entry

https://www.uniprot.org/uniprotkb/P68871/entry

In the UniProt database, there are 819 variants of the hemoglobin subunit α and 771 variants of the hemoglobin subunit β (as of 7^th August 2024, Figure 2). Assuming homozygotes, there are 631,749 potential combinations of hemoglobin. However, the genetic diversity increases exponentially when considering heterozygotes, resulting in over 10¹¹ possible combinations. This number surpasses the current human population on Earth, illustrating the astonishing potential variations in known combinations of hemoglobin variants. The actual diversity is likely much lower because Hb variants are often present in specific geographically constrained sub-populations. For example, HbS is prevalent in Africa and India, thalassemias are common in the Mediterranean, Middle East, and Southeast Asia, HbC is common in West Africa, HbD-Punjab has been found in the Indian subcontinent, HbE is highly prevalent in Southeast Asia, Hb Lepore is prevalent in Southern Italy and Mediterranean populations, Hb Constant Spring (Hb CS) is common in Southeast Asia, Hb Bart's (γ4) has been found in individuals with α-thalassemia, prevalent in Saudi Arabia and Thailand.

The classification of genetic variants, particularly in hemoglobinopathies, is crucial for accurate diagnosis and clinical management. The American College of Medical Genetics and Genomics (ACMG) guidelines provide a five-category system: pathogenic, likely pathogenic, uncertain significance, likely benign, and benign [31,32]. However, this system has limitations in addressing the spectrum of variant effects. An expanded seven-category system, including "predisposing" and "likely predisposing," has been proposed to better classify variants in disease-causing genes [31,33]. Variant reclassification, driven by new evidence, can significantly impact patient care, with reclassification rates ranging from 3.6% to 58.8% [34]. For hemoglobinopathies, molecular genetic testing is essential for accurate diagnosis and genetic counseling [35,36]. The clinical implications of variant classification are profound, affecting organ surveillance, prophylactic surgery, and cascade testing [37].

Hemoglobinopathies are a group of disorders affecting the hemoglobin molecule. These disorders typically result from genetic mutations that lead to abnormal structure or production of hemoglobin, causing various health problems ranging from mild anemia to severe complications. For example, HbM variants lead to methemoglobinemia, where iron in the heme group is oxidized to the ferric form, impairing oxygen delivery. Methemoglobinemia is a blood disorder characterized by elevated levels of methemoglobin, which impairs oxygen delivery to tissues [38]. It can be inherited or acquired, with the latter being more common and often caused by medications or chemicals [39]. Symptoms include cyanosis unresponsive to oxygen therapy and chocolate-colored blood [40]. Diagnosis is confirmed through co-oximetry, arterial blood gas analysis, and serum methemoglobin levels [41]. Treatment involves removing the offending agent, administering oxygen, and using methylene blue as an antidote in severe cases [42]. Dapsone is a common cause of acquired methemoglobinemia, while benzocaine spray has been associated with severe cases [43]. In pediatric patients, underlying hematologic diseases and G6PD deficiency can predispose to methemoglobinemia [44]. Increased awareness and primary prevention efforts are crucial to reduce morbidity and mortality associated with this condition [43].

Sickle cell disease (SCD) is a genetic disorder caused by a single nucleotide mutation in the β-globin gene, resulting in the production of abnormal hemoglobin S (HbS) [45,46]. This E6V mutation leads to HbS polymerization under low oxygen conditions, causing red blood cell (RBC) sickling and hemolysis [45,47]. SCD is characterized by chronic inflammation, oxidative stress, and vascular dysfunction, contributing to various complications such as stroke, pulmonary hypertension, and renal disorders [46,48]. The disease affects millions worldwide, with higher prevalence in Africa and India [47]. Recent advancements in care, including newborn screening, penicillin prophylaxis, and hydroxyurea treatment, have improved patient outcomes [49,50]. Ongoing research explores gene editing techniques like CRISPR/Cas9 to target the β-globin gene in hematopoietic stem cells as a potential therapeutic approach [51]. Gene therapy approaches using βT87Q-globin have shown anti-sickling effects by reducing endogenous βS-globin expression [52]. These advancements in antisickling agents and therapies offer new possibilities for treating sickle cell disease.

Thalassemias are inherited disorders of hemoglobin synthesis, characterized by deficient production of globin chains [53,54]. They are prevalent in Mediterranean, Middle Eastern, and Southeast Asian regions [55,56]. Alpha and beta thalassemias are the most common types, resulting from mutations in α and β globin genes, respectively [57]. Symptoms range from asymptomatic in carriers to severe anemia requiring regular blood transfusions in major forms [58]. Complications include iron overload, cardiac issues, and endocrine disorders[59]. Treatment options include blood transfusions, iron chelation therapy, and bone marrow transplantation [55]. Gene therapy and editing strategies are emerging as potential curative treatments [53]. Prevention through premarital screening and prenatal diagnosis is crucial in reducing the prevalence of thalassemia [59]. Despite its significant impact, thalassemia is often underrecognized in global health assessments [54].

Hemoglobin C (HbC) is a common structural variant of normal hemoglobin, resulting from a mutation in the beta-globin gene [60]. HbC is less soluble than normal hemoglobin, leading to increased rigidity of red blood cells [61]. This can cause mild chronic hemolysis, splenomegaly, and jaundice in homozygous individuals [60]. HbC interacts more strongly with erythrocyte membranes than normal hemoglobin [62]. While HbC disease is generally mild, its inheritance with other hemoglobinopathies like HbS can have serious consequences [60]. Both HbC and HbS may affect the development of acquired immunity against malaria [63]. Efforts have been made to develop simple, inexpensive methods for detecting HbC in resource-limited settings, including microscopic visualization of HbC crystals [64,65]. These methods could potentially determine zygosity and aid in diagnosis in underdeveloped countries where HbC is prevalent.

Hemoglobin D-Punjab (HbD-Punjab) is a variant resulting from a mutation in codon 121 of the β-globin gene [66]. While heterozygous HbD-Punjab is generally asymptomatic, homozygous cases can cause mild hemolytic anemia and splenomegaly [66,67]. The clinical presentation of HbD-Punjab can vary, especially when coinherited with other hemoglobinopathies or thalassemias [68,69]. HbSD-Punjab, a compound heterozygous condition, can lead to severe symptoms resembling sickle cell disease [70]. HbD-Punjab/β-thalassemia combinations may result in moderate to severe anemia [67,71,72]. Diagnosis typically involves hemoglobin electrophoresis and molecular studies [68]. Treatment options include blood transfusions and hydroxyurea, which has shown promise in managing symptoms [70]. The prevalence of HbD-Punjab is relatively low, estimated at 0.06% in one study [66].

Hemoglobin E (HbE), a common β-globin variant in Southeast Asia, results from a Glu26Lys mutation [73,74]. This variant exhibits extended linkage disequilibrium and likely arose 1,240-4,440 years ago due to malarial selection [73]. HbE frequencies reach up to 0.433 in some Lao populations [74]. While HbE shows minimal allosteric changes, it demonstrates altered redox properties, including decreased nitrite reductase activity and accelerated reduction by cysteine [75]. HbE is more susceptible to oxidative damage, especially in the presence of free α subunits [76]. When co-inherited with β-thalassemia, HbE can cause severe clinical manifestations [77]. However, its association with protection against cerebral malaria remains inconclusive [78]. HbE disorders present a wide range of clinical severity, from asymptomatic carriers to severe anemia [79].

Hemoglobin [80]Bart's (γ4) is a homotetrameric hemoglobin found in individuals with α-thalassemia [81]. It lacks cooperativity and has a higher oxygen affinity than adult or fetal hemoglobin. Crystal structures reveal that Hb Bart's resembles the R state of adult hemoglobin [81,82]. Quantification of Hb Bart's in cord blood accurately indicates α-thalassemia status, with levels correlating to the number of deleted or inactivated α-globin genes [83,84]. The prevalence of Hb Bart's varies among populations, with high rates reported in Saudi Arabia and Thailand [85,86]. Spectroscopic studies show that Hb Bart's is structurally similar to Hb H (β4) [87]. In some populations, elevated Hb Bart's may result from developmental asynchrony rather than α-thalassemia [88].

Hemoglobin Chesapeake (α92 Arg→Leu) is a high-oxygen-affinity hemoglobin variant associated with erythrocytosis [89,90]. This mutation occurs at the α1β2 interface, similar to other high-affinity variants like Hb Wood and Hb Malmö [91]. The increased oxygen affinity results from an early conversion from the T to R state during ligand binding [90]. Hb Chesapeake causes mild erythrocytosis in carriers due to reduced oxygen delivery to tissues, necessitating increased red cell production [92]. This mechanism is also observed in other high-affinity variants like Hb Kempsey [93]. The fetal form of Hb Chesapeake (α2Chesγ2) also exhibits increased oxygen affinity, suggesting similar conformational changes in the γ chain [94]. Generally, high-affinity hemoglobin variants impair the formation of a stable T state or modify the heme environment, leading to compensatory erythrocytosis [95].

Hemoglobin Lepore is a structural variant formed by the fusion of δ and β globin genes, resulting from unequal crossover events [96,97]. It is associated with a β-thalassemia minor phenotype in carriers and can lead to severe anemia in homozygotes or compound heterozygotes with β-thalassemia [98,99]. Several variants exist, including Lepore-Boston, Lepore-Hollandia, and Lepore-Washington-Boston [100,101]. The condition is prevalent in Southern Italy and has spread globally due to migration [99]. Diagnosis requires hematological, biochemical, and molecular analyses [96]. Hb Lepore can interact with other hemoglobinopathies, resulting in various clinical phenotypes [101]. Recently, a novel variant, Hb Lepore-Hong Kong, was identified in a Chinese family [102]. Accurate diagnosis is crucial for genetic counseling and prenatal screening in affected populations.

Hemoglobin Constant Spring (Hb CS) is a variant caused by a mutation in the α2-globin gene termination codon, resulting in an elongated α-chain [103]. It is prevalent in Southeast Asian populations, with gene frequencies ranging from 0.008 in Thailand to 0.143 in Vietnam [104,105]. Hb CS can lead to thalassemia intermedia when combined with α-thalassemia [106]. Diagnosis is challenging due to low amounts of the mutant hemoglobin, but selective enzymatic amplification of α2-globin DNA allows for accurate detection [107]. Hb CS can interfere with glycated hemoglobin measurements using boronate affinity chromatography [108]. In rare cases, homozygous Hb CS can cause fetal anemia and hydrops [109]. Understanding Hb CS is crucial for genetic counseling and prenatal diagnosis in affected populations. Hb O-Arab (Glu121Lys) causes mild hemolytic anemia in populations from the Middle East and North Africa.

Hemoglobin O Arab is a rare abnormal hemoglobin variant characterized by a β121Glu → Lys mutation [110,111]. It can occur in homozygous form or in combination with other hemoglobinopathies. The homozygous form is generally well-tolerated [112,113], while compound heterozygous forms, particularly Hb S/O Arab, can result in severe clinical manifestations like sickle cell anemia [110,114]. Hb S/O Arab patients may experience acute chest syndrome, vasoocclusive events, and other complications [110]. Diagnosis requires specific electrophoresis techniques due to Hb O Arab's migration patterns [110]. The condition has been reported in various populations, including African Americans, West Africans, and Arabs [110,112,115]. Treatment may involve transfusions and splenectomy in cases of hypersplenism [113].

Hb Seal Rock, an extended α-chain variant, is associated with mild Hb H disease and α-thalassemia-2 trait [116]. Some α-chain variants can lead to chronic hemolytic anemia, with the mutation's location within the gene sequence playing a crucial role in determining its effect [117]. Hb Indianapolis, a rare and slightly unstable β-globin variant, was reported to cause moderate hemolytic anemia and renal damage in a Brazilian patient [118]. Routine DNA sequencing of α- and β-globin genes has revealed numerous novel mutations, including 11 new β-chain variants, 15 α-chain variants, 19 β-thalassemia mutations, and 15 α+-thalassemia mutations, highlighting the genetic diversity in hemoglobinopathies [119].

Several unstable hemoglobin variants have been identified that cause mild to moderate hemolytic anemia. Hb Madrid (β115Ala→Pro) was found in a Spanish boy and a Korean family, resulting in moderately severe hemolytic anemia [120,121]. Hb Showa-Yakushiji (β110Leu→Pro) was observed in four unrelated Indian patients [122]. Other variants include Hb Santander (β34Val→Asp) in a Spanish patient [123], Hb Yokohama (β31Leu→Pro) in a Japanese family [124], and Hb Seattle (β76Ala→Glu) in a mother and her two sons [125]. Hb Miami (β116His→Pro) and Hb Hershey (β70Ala→Gly) were found in association with beta-thalassemia mutations, while Hb Abington (β70Ala→Pro) was another unstable variant [126]. These variants demonstrate the range of clinical severity observed with unstable hemoglobin mutations, particularly when combined with thalassemic mutations.

2.2. Hemoglobin Instability and Heinz Bodies

Hemoglobin instability can lead to denaturation and precipitation, resulting in the formation of Heinz bodies, which are inclusions of denatured hemoglobin in red blood cells. This instability is often due to amino acid substitutions or deletions that disrupt the hemoglobin structure, enhancing oxidation to methemoglobin and subsequent conversion to hemichrome [127]. The loss of heme, particularly from beta chains, is a critical factor in Heinz body formation [128,129]. The presence of Heinz bodies can cause hemolytic anemia due to mechanical obstruction in microcirculation and oxidative damage to red cell membranes [80,130]. Additionally, factors such as temperature and oxidative stress can exacerbate the formation of Heinz bodies [131]. Oxidative stress occurs when there is an imbalance between reactive oxygen species (ROS) and the body's antioxidant defenses. Hemoglobin is particularly vulnerable to oxidative damage, which can lead to hemolysis. This oxidative damage is exacerbated in conditions such as thalassemia and sickle cell disease. Mechanisms leading to Heinz body formation include oxidative damage, genetic mutations, chemical exposure, and enzyme deficiencies. Reactive oxygen species (ROS) can oxidize hemoglobin, causing it to denature and form Heinz bodies. This oxidative stress is common in conditions with an elevated level of ROS, such as inflammation or chronic diseases.

As mentioned above genetic factors significantly influence the stability of hemoglobin. Mutations in the genes encoding hemoglobin can lead to structurally unstable hemoglobin variants, making them more susceptible to denaturation and resulting in disorders such as thalassemias and hemoglobinopathies. These genetic mutations can affect the function and stability of hemoglobin molecules, leading to various clinical manifestations. Out of the 819 known variants of the hemoglobin subunit α, 31 are classified as unstable. This represents approximately 3.8% of all α subunit variants. In contrast, 85 out of 771 known variants of the hemoglobin subunit β are considered unstable, accounting for about 11% of the β subunit variants. This comparison highlights a significant difference in the frequency of unstable variants between the two subunits. The frequency of unstable variants in the β subunit is nearly three times higher than that in the α subunit, suggesting that the β-globin gene may be more susceptible to mutations that result in instability or that the structural constraints of the β subunit allow for more potentially unstable configurations.

The human α-globin gene cluster, including HBA1 and HBA2, is located on chromosome 16 at position 16p13.2-pter [132,133]. This region is extremely gene-rich, containing 100 confirmed and 20 predicted genes [134]. The α-globin locus is highly polymorphic, with 15 dimorphic and 2 multiallelic genetic markers identified [135]. Upstream regulatory elements, particularly HS-40, play crucial roles in α-globin gene expression [136]. α-Thalassemia, a disorder of reduced α-chain synthesis, is often caused by large deletions within the gene cluster [137]. Interestingly, a 62 kb deletion upstream of the α-globin genes can also cause α-thalassemia without directly affecting the genes themselves [138]. The α-globin locus serves as an ideal genetic marker for constructing human linkage maps and studying molecular genetics of the cluster [135].

The β-globin gene cluster, located on chromosome 11 at position 11p15.5, includes the HBB gene, which encodes the β-globin chain of adult hemoglobin [139,140]. This cluster comprises several genes, including HBD, which encodes the δ-globin chain, and undergoes critical expression switches during development [141]. The precise localization of the β-globin gene has been confirmed through various studies, indicating its location within the 11p15.4 to 11p15.5 region [142,143,144]. The regulation of these genes is crucial for understanding hemoglobinopathies, with recent findings highlighting the role of factors like BCL11A in silencing fetal hemoglobin [145]. Overall, the β-globin gene cluster is essential for normal hemoglobin function and is implicated in disorders such as sickle cell anemia and β-thalassemia [140].

The observed difference in the frequency of unstable variants between the α and β subunits, 3.8% vs. 11%, may be due to several factors. One would expect that the α-globin gene cluster location on chromosome 16 and its gene duplication may confer additional stability, resulting in higher chance for accepting unstable variants compared to the β-globin gene cluster on chromosome 11. The presence of two nearly identical genes (HBA1 and HBA2) in the α-globin cluster means there is a redundancy that can potentially compensate for potential mutations. If one gene is mutated, the other can often continue to produce functional α-globin chains, reducing the impact of any single mutation and enhancing overall stability. This redundancy might act as a genetic buffer, making the α-globin production more resilient to genetic mutations and environmental pressures. However, as we found that less unstable variants of α-chain are nearly three-fold less indicating that other factors are responsible for higher number of unstable β-chain variants. Other factor would be structural differences between the α and β subunits might inherently affect their susceptibility to mutations that lead to instability. While both subunits share a high sequence identity of 43% and 50-60% sequence similarity, they may have distinct tolerance toward mutations constrained by their differences in function

The β-globin's versus α-globin's specific role and interactions in the hemoglobin tetramer may be reason for higher counts of unstable mutations. In fact, the β-globin plays a crucial role in hemoglobin structure and function. It forms strong interactions with α-globin to create stable α1β1 dimers, which then assemble into α2β2 tetramers [146]. The β-chains significantly influence oxygen binding characteristics, particularly through alterations in the T state properties [147]. The β112 Cys residue is critical for both homo- (β4) and hetero-tetramer (α2β2) formation, with mutations at this position affecting assembly and stability [148]. The β4 tetramer structure closely resembles the R state of liganded α2β2 hemoglobin, but with some unique interface interactions [149]. The β chains also interact with the cytoplasmic domain of band 3 in erythrocyte membranes, preferentially binding to deoxyhemoglobin [150]. On the other hand, α-globin plays a crucial role in hemoglobin formation and function. The α-Hemoglobin Stabilizing Protein (AHSP) is essential for maintaining α-globin stability and preventing its precipitation [151]. AHSP binds specifically to the G and H helices of α-globin, with the N-terminal part of the H helix being critical for this interaction[152,153]. AHSP acts as a molecular chaperone, facilitating α-globin folding, refolding after denaturation, and incorporation into hemoglobin A[154]. It also protects against oxidative damage and helps maintain the correct ratios of α and β globins during hemoglobin biosynthesis [155]. Mutations in α-globin that impair AHSP binding can lead to protein instability and anemia [156]. The complex interplay between α-globin expression, AHSP, and hemoglobin assembly is crucial for normal erythropoiesis [157]. Hence, we can speculate that these differences between globins may result in observed differences in frequencies of unstable variants that α-globin mutations produce a significant disadvantage, so they are much less tolerated than β-globin unstable variants. In addition, a possible correlation can be found between amino acid residues contacting heme and unstable variants (Fig. 2d).

2.3. Pharmacological Approaches to Hemoglobin Stabilization and Oxidative Stress in Hemolytic Disorders

Certain molecules and toxins can induce oxidative stress or directly interact with hemoglobin, leading to its denaturation and the formation of so-called Heinz bodies. Even below mentioned small molecules are not directly interacting with Hb, they can influence metabolism of erythrocytes, reduce oxidative stress, or indue other hemoglobin chain expression and by doing this they can provide stability for hemoglobin or prevent sickling.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting approximately 400 million people worldwide, is a crucial enzyme in protecting red blood cells from oxidative stress [158]. G6PD deficiency can lead to hemolytic anemia, neonatal jaundice, and increased susceptibility to oxidative damage [159]. The deficiency results in reduced NADPH production, compromising the cell's ability to maintain glutathione in its reduced state [160]. This leads to increased hemoglobin denaturation and ferriheme release, potentially causing hemolysis [161]. Contrary to previous beliefs, NADPH status, rather than glutathione levels, modulates oxidant sensitivity in G6PD-deficient erythrocytes [162]. G6PD deficiency also has broader implications, including increased risk of prenatal and postnatal death, infertility, and teratogenesis [163]. In transfusion medicine, G6PD-deficient blood may pose risks to certain patient populations [164].

Oxidative stress plays a significant role in hemolytic anemias, causing damage to red blood cells and exacerbating symptoms [165]. Antioxidants, such as N-acetylcysteine (NAC) and Vitamin E, have shown promise in reducing oxidative stress and improving hemoglobin levels, particularly in children with transfusion-dependent thalassemia [166]. These antioxidants can inhibit cation pathways responsible for red blood cell dehydration and reduce phosphatidylserine exposure, potentially improving cell rheology and reducing vascular adhesion [167]. Supplementation with vitamin C and NAC has been found to preserve glutathione homeostasis and reduce hemolysis in stored red blood cells [168]. However, high-dose vitamin C and E supplementation may worsen hemolysis in some cases [169]. Combining antioxidants with iron chelators may provide substantial improvements in the pathophysiology of hemolytic anemias, especially thalassemia [170].

Hemin is a pharmacological agent used in treating acute porphyrias, which are metabolic disorders characterized by heme biosynthesis defects. It functions primarily by inhibiting delta-aminolevulinic acid synthase (ALAS-1), the rate-limiting enzyme in heme production, thereby reducing the accumulation of toxic heme precursors like delta-aminolevulinic acid [171,172]. Hemin administration has been shown to alleviate symptoms during acute attacks by restoring heme levels and downregulating ALAS-1 activity [173,174]. Additionally, heme's feedback inhibition on ALAS-1 is mediated through various mechanisms, including proteolytic degradation of the enzyme [175]. The clinical efficacy of hemin underscores its role in managing acute porphyrias, as it directly addresses the biochemical disturbances underlying these conditions [176,177].

Hydroxyurea is a promising treatment for sickle cell disease, primarily due to its ability to induce fetal hemoglobin (HbF) production [178,179,180]. HbF inhibits the polymerization of sickle hemoglobin (HbS), reducing disease severity [179]. Hydroxyurea works by perturbing erythroid precursor maturation and potentially through nitric oxide-dependent mechanisms [181]. Clinical studies have shown that hydroxyurea treatment increases HbF levels, reduces hemolysis, and decreases the frequency of vaso-occlusive crises and hospitalizations [182,183]. The drug also improves red cell survival and reduces sickling at partial oxygen saturation [184]. While hydroxyurea's effects can be inconsistent, it has been shown to reduce morbidity and mortality in adults with sickle cell anemia [178]. Ongoing research is exploring its use in children and in combination with other HbF-inducing agents [179,185].

Mitapivat, an oral pyruvate kinase activator, has shown promising results in treating various hemoglobinopathies. In pyruvate kinase deficiency, it increased hemoglobin levels and reduced transfusion burden, with sustained long-term effects [186,187]. For non-transfusion-dependent α- and β-thalassemia, mitapivat improved hemoglobin levels, markers of erythropoiesis, and hemolysis [188,189,190]. In sickle cell disease, it demonstrated improvements in anemia, hemolysis, and hemoglobin S polymerization kinetics [191]. Mitapivat's mechanism of action involves increasing ATP production in red blood cells, which may help stabilize hemoglobin and improve cell integrity [192]. The drug has shown a favorable safety profile across studies, with common adverse events being generally mild and transient [187,188]. These findings suggest that mitapivat may represent a novel therapeutic approach for various hereditary anemias [193].

2.4. Small-Molecule Binders to Hemoglobin

Hemoglobin is also a direct drug target and hence several classes of molecules were found to bind and stabilize hemoglobin, which should improve oxygen delivery in patients with hemoglobinopathies such as sickle cell disease and β-thalassemia. Pharmacological chaperones are drugs that stabilize hemoglobin by promoting proper folding and preventing denaturation. These agents help maintain hemoglobin's functional state under stress conditions, reducing the likelihood of hemolysis (see above). Most of the development of drugs targeting hemoglobin focus on sickle cell diseases, which is caused by a single mutation that results in the substitution of valine for glutamic acid in the sixth position of the β-globin chain of hemoglobin, leading to the polymerization of hemoglobin S upon deoxygenation. Peptides and amino acids stabilizing hemoglobin have been summarized in detail in a recent review [194]. Recent advances in SCD treatment have expanded beyond the traditional therapies of hydroxyurea and L-glutamine. New approaches target various aspects of SCD pathophysiology, including HbS polymerization, cellular adhesion, inflammation, and oxidative stress [195]. The FDA has approved three new drugs: crizanlizumab, voxelotor, and L-glutamine, which have shown efficacy in reducing pain crises and improving hemoglobin levels [196]. Emerging therapies include peptide-based inhibitors of HbS polymerization, which represent a novel approach to SCD treatment [194]. Additionally, researchers are exploring combination therapies to address the complex nature of SCD manifestations [197,198]. While gene therapy holds promise for a cure, its widespread application remains limited due to technical and cost challenges [199]. These developments offer hope for improved quality of life and survival for SCD patients.

Compounds like TD7, VZHE039, and their nitric oxide-releasing derivatives have shown promising antisickling properties by increasing hemoglobin's oxygen affinity and disrupting HbS polymerization [200]. TD-1, a small molecule identified through screening, demonstrated dual allosteric and antioxidant effects by binding to βCys93 and βCys112, stabilizing hemoglobin's relaxed state and reducing oxidative changes [201,202]. Other compounds like TD-3 and hydroxyurea also target βCys93, providing both antioxidant and antisickling benefits [203]. These studies have elucidated the mechanisms of action for various antisickling agents, including their effects on oxygen affinity, HbS polymerization, and oxidative stress reduction. Further research on antisickling agents targeting hemoglobin's β-Cys93 has shown promising results for treating sickle cell disease. Other thiol reagents reacting with β-Cys93 have shown varying antisickling effects [204,205] Overall, increasing hemoglobin's oxygen affinity represents a promising therapeutic approach for sickle cell disease.

5-hydroxymethyl-2-furfural (5HMF) is a promising compound for treating sickle cell disease. It forms a Schiff-base adduct with hemoglobin, increasing oxygen affinity and inhibiting red blood cell sickling [206,207]. 5HMF demonstrates dose-dependent effects on hemoglobin oxygen affinity in phase 1 clinical trials [208]. It improves cardiac function during severe hypoxia [209] and reduces deoxygenation-induced dehydration of red blood cells by inhibiting cation conductance and Ca2+-activated K+ channels [210]. Researchers have developed ester and ether derivatives of 5HMF with enhanced antisickling properties [207] and explored nitric oxide-releasing prodrugs [211]. The mechanism of action involves stabilizing the R state of hemoglobin, which has higher oxygen affinity and slower polymerization kinetics [212,213]. These findings support 5HMF as a potential therapeutic agent for sickle cell disease.

Hb plays a crucial role in nitric oxide metabolism and oxygen transport. Hb can form various NO-related compounds, including nitrosylated Hb (HbNO) and S-nitrosohemoglobin (SNO-Hb) [214,215]. These compounds are involved in regulating vascular function and oxygen delivery [216,217]. The interaction between Hb and NO is complex, involving redox reactions and allosteric effects [214,218]. SNO-Hb has been proposed as a key mediator of NO transport and bioactivity, with its formation and release coupled to oxygen saturation [216,219]. However, the precise mechanisms of NO activity preservation and regulation by Hb remain debated [220,221]. Understanding these interactions is crucial for developing transfusion therapeutics and treating various cardiovascular diseases [217].

Recent research on SCD has also focused on developing aromatic aldehydes as potential treatments by increasing hemoglobin oxygen affinity and inhibiting red blood cell sickling. Novel compounds, including pyridyl derivatives of vanillin and azolylacryloyl derivatives, have shown improved potency, metabolic stability, and sustained antisickling effects in vitro [222,223]. Some compounds exhibit both oxygen-dependent and oxygen-independent antisickling mechanisms, potentially disrupting key intermolecular contacts necessary for hemoglobin S polymer formation. Crystal structure studies have provided insights into the binding modes and mechanisms of action of these compounds [223]. The development of hemoglobin modulators represents a promising approach for treating SCD, with ongoing research and growing intellectual property in this field [224]. These advancements complement other therapeutic strategies, including targeting cellular adhesion, inflammation, and oxidant injury.

Figure 3. Binding interactions of various small molecules with different Hb residues as visualized in their respective PDB structures. The figure shows the chemical structures of the ligands (such as toluene, 1H-1,2,3-triazole-5-thiol, inositol hexakisphosphate, and others) along with their binding pockets and the chains they interact with, as indicated by the PDB IDs (4NIO, 6BWU, 3HXN, etc.). The protein-ligand interactions are depicted through dashed lines, highlighting key residues involved in the binding. Residues interacting with hemoglobin chains (e.g. A, B, C) are listed. .

Figure 3. Binding interactions of various small molecules with different Hb residues as visualized in their respective PDB structures. The figure shows the chemical structures of the ligands (such as toluene, 1H-1,2,3-triazole-5-thiol, inositol hexakisphosphate, and others) along with their binding pockets and the chains they interact with, as indicated by the PDB IDs (4NIO, 6BWU, 3HXN, etc.). The protein-ligand interactions are depicted through dashed lines, highlighting key residues involved in the binding. Residues interacting with hemoglobin chains (e.g. A, B, C) are listed. .

PF-07059013 (compound 23) is a noncovalent hemoglobin modulator that has advanced to phase 1 clinical trials for sickle cell disease (SCD) treatment. It demonstrated a 37.8% reduction in red blood cell sickling in a mouse model [225]. This compound represents a new approach to SCD treatment, targeting the root cause of the disease: hemoglobin S polymerization [226]. Unlike earlier covalent modifiers with potential safety concerns, PF-07059013 binds noncovalently to hemoglobin [227]. Similar approaches, such as GBT440, have shown promise in preclinical testing [228,229]. These developments are part of a broader effort to create new SCD therapies, including fetal hemoglobin inducers and agents targeting cellular adhesion, inflammation, and vascular tone [230]. Such advancements offer hope for improved SCD management beyond current treatments like hydroxyurea and blood transfusions [231].

Vanillin derivatives have shown promise as potential treatments for sickle cell disease and other conditions. Novel compounds like SAJ-009, SAJ-310, and SAJ-270 demonstrated improved binding and pharmacokinetic properties compared to vanillin, with enhanced allosteric and antisickling effects [223]. SAJ-310 exhibited sustained antisickling activity and potential metabolic stability [232]. Vanillin derivatives also showed antioxidant properties and inhibitory activity against acetylcholinesterase and β-amyloid aggregation, suggesting potential applications in Alzheimer's disease treatment [233]. Additionally, vanillin demonstrated analgesic effects on mechanical allodynia in a neuropathic pain model [234] and inhibitory activity against mushroom tyrosinase [235]. The compound's ability to react covalently with sickle hemoglobin and inhibit cell sickling supports its potential as a treatment for sickle cell anemia [236]. Vanillin's diverse pharmacological activities and safety profile make it a promising candidate for further research and development.

VZHE-039 is a novel antisickling agent that exhibits both oxygen-dependent and oxygen-independent mechanisms of action [237]. In this study, VZHE-039 demonstrates enhanced pharmacologic activities and metabolic stability. Its oxygen-independent activity is attributed to interactions with the αF-helix of hemoglobin, potentially destabilizing the sickle hemoglobin polymer [238]. Other antisickling agents, such as voxelotor and FT-4202, also show promise in treating sickle cell disease by increasing hemoglobin oxygen affinity.

Crown ethers, cyclic polyethers with oxygen atoms, have shown potential in modulating protein surface properties and interactions [239]. They can bind to metal ions and positively charged amino acid side chains, affecting protein behavior such as oligomerization and crystallization. In hemoglobin research, crown ethers and related compounds have been explored for their ability to modify hemoglobin's oxygen-binding properties. Acylation of hemoglobin with polyoxyethylene derivatives has been shown to alter its oxygen affinity [240]. Other synthetic allosteric effectors, such as RSR-4 and RSR-13, have demonstrated the ability to shift hemoglobin's allosteric equilibrium towards the low-affinity T-state [241]. These studies highlight the potential of crown ethers and related compounds in modulating hemoglobin function and developing novel therapeutic approaches.

Complex interactions between hemoglobin (Hb) can occur also with natural molecules such as including sphingosine 1-phosphate (S1P) and lipopolysaccharide (LPS). S1P is a bioactive lipid involved in immune responses, cell growth, and survival. It has been found to bind specifically to deoxygenated sickle hemoglobin (HbS), the form of hemoglobin that promotes the sickling of red blood cells in sickle cell disease. When S1P binds to HbS, it alters glucose metabolism and increases oxidative stress, both of which are significant contributors to the pathology of sickle cell disease. Altered glucose metabolism disrupts energy production in red blood cells, while increased oxidative stress leads to cellular damage and inflammation, further exacerbating the disease's symptoms [242]. Hb also interacts with lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, enhancing its biological activity and potentially contributing to the immune defence against bacterial infections. This interaction suggests that hemoglobin may play a role beyond oxygen transport, acting as a modulator of the immune response [243,244]. Multiple binding sites for LPS have been identified on both the α and β-subunits of hemoglobin, highlighting the molecule's multifunctional nature and its potential importance in pathogen defense [244,245]. Hemoglobin also binds to erythrocyte membranes, an interaction crucial for the stability and integrity of red blood cells. This binding is driven by electrostatic forces influenced by factors such as pH and ionic strength, which may vary under different physiological conditions, such as during oxygenation/deoxygenation cycles [246,247]. A critical protein involved in this interaction is band 3 protein, a major structural component of the erythrocyte membrane. The acidic N-terminal segment of the cytoplasmic domain of band 3 serves as a primary binding site for hemoglobin [150]. This interaction is essential for maintaining the structural integrity of red blood cells and regulating their lifespan. Disruptions in this binding can lead to membrane instability and hemolysis, a hallmark of hemolytic anemias such as sickle cell disease. In pathological conditions like sickle cell disease, these hemoglobin interactions are further altered, contributing to disease progression. The S1P-Hb interaction exacerbates oxidative stress, worsening the symptoms of sickle cell disease. The LPS-Hb interaction could play a dual role in immune defense and the potential triggering of exaggerated immune responses. Finally, hemoglobin's binding to the erythrocyte membrane via band 3 protein is critical for cell stability, and disruptions in this interaction could contribute to the shortened lifespan of red blood cells seen in sickle cell disease and other hemoglobinopathies. In summary, these complex interactions between hemoglobin, S1P, LPS, and the erythrocyte membrane are critical for both normal physiological processes and pathological conditions, especially sickle cell disease.

Voxelotor is a novel oral treatment for SCD that binds to hemoglobin, increasing its oxygen affinity and preventing polymerization of sickle hemoglobin (HbS) [248,249]. By forming a reversible covalent bond with hemoglobin, voxelotor reduces sickling of red blood cells and interrupts the molecular pathogenesis of SCD. Clinical trials have demonstrated that voxelotor increases hemoglobin concentration, reduces hemolysis, and improves hematological parameters in SCD patients [250,251]. The drug has shown a dose-dependent pharmacokinetic and pharmacodynamic response and is well-tolerated [252]. Studies in SCD mice have indicated that increasing hemoglobin oxygen affinity with voxelotor analogs improves brain oxygenation and hypoxia tolerance (Dufu et al., 2021). Voxelotor represents a new disease-modifying approach to SCD treatment, targeting the root cause of the disease [253].

3. Search for Potentially New Hb Stabilisers: Hemoglobin Binding Proteins

Innovative approaches targeting hemoglobin stability and function may enhance protein platforms for protein engineering by the use of protein chaperones, enzymes like falcilysin, and nanobodies.

Alpha-hemoglobin-stabilizing protein (AHSP) is an erythroid-specific molecular chaperone that binds and stabilizes free α-globin chains, preventing their precipitation and cytotoxic effects [254,255]. AHSP forms a 1:1 complex with α-globin, binding to its G and H helices in a manner similar to the α1-β1 interface in hemoglobin A [153,256]. This interaction facilitates the conversion of oxy-αHb to a non-reactive, oxidized form, limiting its ability to generate reactive oxygen species [257]. AHSP plays a crucial role in normal erythropoiesis and hemoglobin synthesis, as demonstrated by studies in AHSP-null mice [154,257]. It also acts as a folding chaperone for nascent α-globin, promoting its incorporation into hemoglobin A. The importance of AHSP in vivo is further supported by mutagenesis studies, which reveal its robust chaperone activity even when its biochemical interactions with α-globin are altered [258].

Falcilysin, a metalloprotease in the malaria parasite Plasmodium falciparum, plays a crucial role in hemoglobin degradation within the acidic food vacuole [259,260]. It operates downstream of aspartic and cysteine proteases, cleaving peptide fragments of hemoglobin rather than intact globin. Interestingly, falcilysin exhibits activity at both acidic and neutral pH, suggesting additional functions beyond hemoglobin catabolism, including potential roles in transit peptide degradation in the apicoplast [261,262]. The enzyme's essentiality for parasite development has led to the exploration of piperazine-based hydroxamic acid inhibitors, which effectively target falcilysin [263]. Overall, falcilysin's multifaceted roles highlight its importance in the parasite's survival and present it as a promising target for antimalarial drug development. Another attractive out-of-box option for falcilysin would be re-programming this protein to stabilise hemoglobin.

Recent research has explored the interactions between hemoglobin (Hb) and various molecules, including nanobodies and lipopolysaccharide (LPS). Nanobodies targeting BCL11A, a repressor of fetal hemoglobin, have been developed for potential therapeutic applications in sickle cell disease and β-thalassemia [264,265]. Studies have also identified LPS-binding sites on Hb, which may play a role in innate immune defense [266], but might be potentially used for drug development.

A recent study explored the use of llama-derived nanobodies, particularly NbE11, to develop sensitive diagnostic tools for detecting human hemoglobin [267]. The study determined the crystal structure of the NbE11-hemoglobin complex, revealing specific interactions primarily with the β-subunit of human hemoglobin. NbE11, a llama-derived nanobody, binds to human hemoglobin (Hb) with moderately high affinity (Kd = 147 nM) and the binding is predominantly driven by favorable enthalpy. These interactions are characterized by high specificity and moderate affinity, driven by favorable enthalpic changes. This specificity allows for the development of nanobody-based assays that can differentiate human hemoglobin from that of other species, reducing false positives in diagnostic applications. Structural differences between human Hb and Hb from other vertebrates at the NbE11 binding interface likely explain the lack of cross-reactivity observed in the NbE11-based sandwich ELISA assay. The incorporation of nanobodies in hemoglobin research highlights their potential as both diagnostic and therapeutic tools. The study showed that NbE11 binds specifically to human hemoglobin without cross-reacting with hemoglobin from other vertebrates, making it suitable for precise diagnostic applications.

The exploration of AHSP, falcilysin, and nanobodies as tools for modulating hemoglobin stability and function presents promising avenues for treating hemoglobin disorders and developing precise diagnostic tools. Additionally, molecularly imprinted polymeric nanoparticles have been developed for specific binding and purification of human Hb [268].

4. Conclusions

The wide range of hemoglobin variants presents challenges in drug development. Although most current drugs focus on SCD, there is potential to develop variant-specific therapies that address the molecular diversity of hemoglobinopathies. Personalized medicine approaches, which tailor treatments based on a patient's specific hemoglobin variant, may offer more precise and effective therapies in the future. Investigating the structural differences between hemoglobin variants and how these influence drug binding and efficacy is essential. Drugs that bind effectively to HbS may not work as well for other variants, such as those found in thalassemia or rare hemoglobinopathies like HbC or HbE. Advances in gene editing, such as CRISPR/Cas9, offer the potential for curative treatments by directly correcting the genetic mutations responsible for hemoglobin variants. However, widespread application remains limited due to technical challenges and high costs.

The relationship between drugs and hemoglobin variants is deeply intertwined with the molecular pathology of hemoglobin disorders. Small-molecule binders, pharmacological chaperones, antisickling agents, and novel therapies like nanobodies are all designed to stabilize hemoglobin or correct the aberrant behavior caused by specific variants. To advance research on hemoglobin variants, time-resolved X-ray powder diffraction, especially utilizing laser-driven X-ray sources, offers the potential to capture subtle dynamic differences at the atomistic level, providing deeper insights into the behavior of hemoglobin variants [269]. Complementing this, the development of 3D-printed in situ crystallization plates, which provide precise control over crystallization in an external electric field, represents a valuable advancement for studying challenging-to-crystallize proteins, including hemoglobin variants [270].

Based on understanding of molecular mechanism of actions, therapies target hemoglobin's functional aspects, such as oxygen affinity, polymerization, and oxidative stress, offering hope for patients with hemoglobinopathies like SCD and thalassemia. Continued research and development of both variant-specific treatments and broader hemoglobin modulators will be critical in improving patient outcomes and addressing the diverse challenges posed by hemoglobinopathies.