Therapeutic Strategies to Alter the Oxygen Affinity of Sickle Hemoglobin




The pathophysiology of sickle cell disease involves the polymerization of sickle hemoglobin in its T state, which develops under low oxygen saturation. One therapeutic strategy is to develop pharmacologic agents to stabilize the R state of hemoglobin, which has higher oxygen affinity and is expected to have slower kinetics of polymerization, potentially delaying the sickling of red cells during circulation. This strategy has stimulated the investigation of aromatic aldehydes, aspirin derivatives, thiols, and isothiocyanates that can stabilize the R state of hemoglobin in vitro. One representative aromatic aldehyde agent, 5-hydoxymethyl-2-furfural, protects sickle cell mice from the effects of hypoxia.


Key points








  • The T state of sickle hemoglobin (HbS) is prone to polymerize, promoting red cell sickling.



  • Stabilizers of the R state of HbS have the potential to directly inhibit sickling.



  • R-state stabilizers also increase the affinity of HbS for oxygen.



  • An R-state stabilizer, 5-hydoxymethyl-2-furfural (also known as Aes-103) is currently in clinical trials.






Oxygen affinity of sickle erythrocytes


The erythrocytes in sickle cell disease have long been known to show decreased oxygen affinity compared with those from healthy volunteers. This property is measured as an increase in the partial pressure of oxygen required to produce 50% oxygen saturation (P 50 ), discussed in further detail later. This decreased oxygen affinity is caused at least in part by increased intracellular concentration of 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes, observed generally in all forms of anemia and considered a compensatory adaption that facilitates oxygen release from red cells to the tissues. 2,3-DPG is a product of anaerobic glycolysis, which has been found in recent years to be regulated in erythrocytes by oxygen-regulated sequestration and inactivation of glycolytic enzymes by the cytoskeletal protein band 3. Among patients with sickle cell disease, P 50 and 2,3-DPG levels vary widely, and more increased levels seem to decrease solubility of sickle hemoglobin (HbS), and to increase red cell sickling under hypoxia, although this has not been confirmed by all investigators. In vitro manipulation of human sickle blood to reduce 2,3-DPG content in red cells also reduces hypoxia-induced sickling in vitro. Preliminary investigation suggests that decreased oxygen affinity of HbS may be associated with greater clinical symptoms, but more investigation is needed to confirm this association. Although decreased oxygen affinity may be adaptive in other anemias, it may be counteradaptive in sickle cell disease because of its promotion of the T state of HbS, which promotes sickling. These effects relate to alterations in the conformation of hemoglobin (Hb).




Oxygen affinity of sickle erythrocytes


The erythrocytes in sickle cell disease have long been known to show decreased oxygen affinity compared with those from healthy volunteers. This property is measured as an increase in the partial pressure of oxygen required to produce 50% oxygen saturation (P 50 ), discussed in further detail later. This decreased oxygen affinity is caused at least in part by increased intracellular concentration of 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes, observed generally in all forms of anemia and considered a compensatory adaption that facilitates oxygen release from red cells to the tissues. 2,3-DPG is a product of anaerobic glycolysis, which has been found in recent years to be regulated in erythrocytes by oxygen-regulated sequestration and inactivation of glycolytic enzymes by the cytoskeletal protein band 3. Among patients with sickle cell disease, P 50 and 2,3-DPG levels vary widely, and more increased levels seem to decrease solubility of sickle hemoglobin (HbS), and to increase red cell sickling under hypoxia, although this has not been confirmed by all investigators. In vitro manipulation of human sickle blood to reduce 2,3-DPG content in red cells also reduces hypoxia-induced sickling in vitro. Preliminary investigation suggests that decreased oxygen affinity of HbS may be associated with greater clinical symptoms, but more investigation is needed to confirm this association. Although decreased oxygen affinity may be adaptive in other anemias, it may be counteradaptive in sickle cell disease because of its promotion of the T state of HbS, which promotes sickling. These effects relate to alterations in the conformation of hemoglobin (Hb).




The allosteric states of Hb and sickle cell disease


Hb has been shown to function in equilibrium between 2 classic states: the tense (T) state, which has low affinity for ligand, and the relaxed (R) state, which has high affinity for ligand. The crystal structure of the T-state (unliganded or deoxygenated) or the R-state (liganded or oxygenated) Hb is each made up of 2 alpha-beta heterodimers (α1β1 and α2β2) arranged around a 2-fold axis of symmetry to form a central water cavity with the alpha cleft and beta cleft defining entries into the cavity ( Fig. 1 ). The T→R allosteric transition is characterized by rotation of the α1β1 dimer relative to the α2β2 dimer, which significantly reshapes the central water cavity, resulting in several differences between the quaternary T and R structures. Most notable is the formation of a larger central water cavity; including the alpha and beta clefts in the quaternary T structure with respect to the quaternary R structure, as well as several different interdimer (α1β2 or α2β1, α1α2 and β1β2) hydrogen bond and/or salt-bridge interactions in the T or R structures that stabilize one state relative to the other. Despite the presence of βVal6 in HbS, normal and HbS molecules have identical quaternary structures.




Fig. 1


Quaternary structure of human Hb. Alpha-1 subunit ( gray ), alpha-2 subunit ( yellow ), beta-1 subunit ( magenta ), and beta-2 subunit ( green ). ( A ) Ribbon diagram showing the 4 Hb subunits arranged around a central water cavity. ( B ) The Hb subunits showing access to the central water cavity via the alpha cleft and beta cleft.


The T and R structures were used to formulate the Monod-Wyman-Changeux and the Koshland-Némethy-Filmer allosteric models and later modified by Perutz with his stereochemical construct. Since then, several R-like or T-like conformations within quaternary T and R states, as well as distinct quaternary relaxed states (R2, R3, RR2, RR3, and so forth) that extend beyond the classic T→R transition have been described and/or incorporated in modern allosteric models. Like the R and T structures, the relaxed structures also show significant differences in the geometry of the central water cavities. Unlike a quarter of a century ago, it is now widely accepted that Hb function involves an ensemble of relaxed Hb states in dynamic equilibrium. One such recent evidence is that aromatic aldehydes that increase the oxygen affinity of HbS do so by binding to quaternary R2 structures and not the quaternary R structures to stabilize the relaxed state. An ongoing study also suggests that thiols increase the oxygen affinity of Hb in part by forming a covalent adduct with βCys93 of both the quaternary R and R3 structures in a manner that should prevent formation of the characteristic T-state salt-bridge interaction between βAsp94 and βHis146 when the Hb transitions to the T state and in so doing shifts the allosteric equilibrium to the relaxed state (Martin K. Safo, unpublished data, 2013). Unless noted otherwise, the R state is used to represent the ensemble of relaxed states.




Hb: a target for drug design


The allosteric equilibrium of Hb is modulated by several endogenous heterotopic effectors, such as 2,3-DPG, and hydrogen ions (H + ); the former bind to the beta cleft and preferentially stabilizes the T state relative to the R state. Stabilization of the R state shifts the oxygen binding curve or oxygen equilibrium curve (OEC) of Hb to the left, producing a high-affinity Hb that more readily binds and holds oxygen ( Fig. 2 ). A shift toward the T state (right shift of the OEC) produces a low-affinity Hb that readily releases oxygen. The degree of shift in the OEC is reported as an increase or decrease in P 50 , the oxygen tension at 50% Hb O 2 saturation, whereas the degree of allosteric character is indicated by the slope of the oxygen binding curve (n 50 ).




Fig. 2


The allosteric mechanism of Hb. ( A ) Oxygen equilibrium (or dissociation) curve of Hb. The normal P 50 value is indicated by dashed lines. The left shift and right shift in the curves ( blue ) are associated with various conditions, including allosteric effectors of Hb (AEHs). ( B ) Modulation of Hb allosteric transition by synthetic AEHs. Stabilization of either the R state or T state ( blue ) has potential therapeutic applications. The R represents all identified relaxed states including R2, R3, RR2, RR3, and the classic R. deoxyHb, deoxygenated Hb; oxyHb, oxygenated Hb; Rx, reaction; S o 2 , oxygen saturation.


Several synthetic allosteric effectors of Hb (AEHs) also bind to the surface, alpha cleft or beta cleft, or the middle of the central water cavity to liganded Hb structure (in the R, R2, or R3 state) and/or unliganded Hb structure (in the T state) (see Fig. 1 ) to either shift the OEC to the left or to the right (see Fig. 2 ). The direction and magnitude of the shift depend on preferential stabilization of one state rather than the other through additional hydrogen bond and/or hydrophobic interactions that prevent the rotation associated with the allosteric transition and/or destabilization of a state by removing intersubunit interactions, which facilitates the allosteric movement. For example, RSR-13 (efaproxiral) and several other aromatic propionate analogues bind to the middle of the central water cavity of Hb ; several angstroms away from the beta cleft where 2,3-DPG and other organic phosphates are known to bind. The binding ties the two dimers together, preferentially stabilizing the T state relative to the R state to lower the affinity of Hb for O 2 and enhance its delivery to tissues in a manner physiologically similar to 2,3-DPG (see Fig. 2 B). These AEHs have potential therapeutic applications in treating ischemia-related cardiovascular diseases, such as angina, myocardial ischemia, stroke, and trauma, for which more O 2 is needed to heal tissue or organs. In contrast, a second class of AEHs, which includes several aromatic aldehydes, bind to Hb to increase its O 2 affinity by preferentially stabilizing the R state relative to the T state (see Fig. 2 B). These compounds are potentially useful for the treatment of sickle cell disease.


The availability of the crystal structures of T and the various relaxed states of Hb have contributed significantly to the design of quaternary state–specific AEHs. Because these compounds bind to locations separate from the substrate (heme pocket) or endogenous 2,3-DPG (beta cleft) binding sites, they are not restricted by the need to generate molecules with higher affinities than the natural ligands; moreover, these AEHs can elicit an effect regardless of the natural ligand concentration. Also, because the allosteric activity of Hb can be modulated to varying degrees, it allows the possibility of tailoring drug activity to the severity of the disease state. Some AEHs bind to the same allosteric sites of Hb, but produce different magnitudes of OEC shift and, in some instances, opposite shift. Based on these observation, a general hypothesis was proposed that the effectors’ ability to shift the allosteric equilibrium (ie, the effectors’ potency and/or direction of shift) is caused not only by where the molecule binds but also by how it interacts with the Hb dimer-dimer interface to stabilize or destabilize that allosteric state.




Development of allosteric modifiers of Hb to treat sickle cell disease


As atomic-level understanding of Hb allosteric property and of the interactions between Hb molecules that contribute to Hb polymerization and formation of pathologic fibers became clear, several classes of compounds (eg, urea derivatives, amino acid derivatives, oligopeptides, carbohydrate derivatives, aromatic alcohols, and acids) were developed, most with the objective of disrupting HbS polymer formation. However, most of these compounds had weak, if any, significant antisickling activity, probably because of weak binding to shallow cavities on the surface of the Hb protein, because such cavities do not exclude water and ions and do not provide the necessary environment for strong hydrophobic interactions.


The realization that the polymerization process is exacerbated by the low O 2 affinity of HbS as a result of unusually high concentration of 2,3-DPG in sickle red blood cells (RBCs) led to another rational approach to treat sickle cell disease by increasing the affinity of HbS for oxygen sufficiently to prevent premature release of oxygen, but not so extensively as to compromise tissue oxygenation. Sunshine and colleagues were among the first to suggest that increasing the O 2 affinity of HbS by 4 mm Hg could lead to therapeutically significant inhibition of intracellular polymerization. This approach was also bolstered by the milder clinical severity observed in patients with sickle cell anemia in whom approximately 20% of the red cell Hb content is expressed as the high-O 2 -affinity fetal Hb (HbF), which is now known to inhibit HbS polymerization. The beginning of the 1970s saw the development of such AEHs, most notably aromatic aldehydes, aspirin derivatives, thiols, and isothiocyanates that form covalent adducts with Hb, modifying the protein’s allosteric property to increase its oxygen affinity. The Klotz group reported several benzaldehydes, including the food additive vanillin ( Fig. 3 ), and showed that these compounds form a Schiff-base interaction with the amino terminus of alpha globin. The interaction is sometimes described as transient covalent, lasting only for a short period of time, because the Schiff base exists in equilibrium between the bound and the free aldehyde. Several isothiocyanates (see Fig. 3 ) that form covalent adducts with Hb have also been reported for their antisickling activities, also by virtue of their ability to increase the oxygen affinity of Hb. The aliphatic isothiocyanates (see Fig. 3 ) bind covalently to beta globin Cys93 to disrupt the native T-state salt-bridge interaction between βAsp94 and βHis146. This binding leads to T-state destabilization, explaining their left-shifting property. Binding to the βCys93 was also suggested to explain the significant increase in the solubility of fully deoxygenated HbS by preventing direct polymer contacts. In contrast, aromatic isothiocyanates (see Fig. 3 ) also react at the amino terminal amine on the alpha chain of Hb and show antisickling activities by increasing the oxygen affinity of HbS, which the investigators suggested to be caused by destabilization of the T state. Although the isothiocyanates seem promising because they can be administered at low doses and less frequently, like other AEHs that form permanent covalent interactions with Hb their lack of specificity could lead to toxicity.




Fig. 3


Structures of synthetic allosteric effectors of Hb.


Peter Goodford’s group was the first to use the classic R structure to design left-shifting aromatic aldehyde-acid AEHs that were postulated to cross-link the two symmetry-related alpha globin subunits via a Schiff-base interaction with the N-terminus of αVal1 of one alpha subunit and a hydrogen-bond interaction with the opposite αVal1 of the second alpha subunit, and stabilize the R state relative to the T state. The study resulted in clinical-tested antisickling aromatic aldehydes that include valeresol (12C79; see Fig. 3 ) and tucaresol (589C80; see Fig. 3 ). A later study by Don Abraham suggested that the left-shifting properties of these agents were the result of 2 molecules (not 1 as proposed by Goodford), each forming a Schiff-base interaction with the N-terminus of the αVal1 nitrogen of the T structure (not the R structure as proposed by Goodford) in manner that destabilizes the T state and left shifts the OEC to the R state. Valeresol underwent human testing, and although potent, was not orally bioavailable, and had a short duration of action of 3 to 4 hours following intravenousadministration. Tucaresol was orally bioavailable with more favorable in vivo human pharmacokinetics than valeresol but caused immune-mediated toxicity in longer-term phase-II studies. Although shown not to bind as designed, the discovery of tucaresol and valeresol indicated proof of principle that the allosteric property of Hb could be altered pharmacologically.


Although Goodford and Abraham had proposed seemingly opposing views of the antisickling mechanism of left-shifting aromatic aldehydes, it was not until several years later that the exact mechanism underlying the antisickling effect of these compounds was identified. Our group, working with aromatic aldehydes (eg, vanillin, furfural, 5-ethyl-2-furfural, and 5-hydoxymethyl-2-furfural [5-HMF]; see Fig. 3 ), showed that these compounds bind to the alpha cleft of both the quaternary R2 structure (and not the classic R structure) and quaternary T structure. Although binding adds to the stability of the R2 structure, it additionally destabilizes the T structure, which shifts the allosteric equilibrium to the R state and increases the oxygen affinity of Hb. In contrast with the R2 structure, the alpha cleft of the R structure is sterically crowded because of the presence of the C-terminal residues αTyr140 and αArg141, thus precluding binding to these effectors. When Peter Goodford proposed his design model, only the classic R and T structures were known. There are several aromatic aldehyde AEHs that also bind to the alpha cleft to form Schiff base with αVal1 nitrogen, but instead of left shifting the OEC, they decrease Hb affinity for oxygen. An ortho-substituted or para-substituted carboxylate moiety (relative to the aldehyde functional group) in these right shifters, such as 5-formylsalicylic acid (see Fig. 3 ) and 2-(benzyloxy)-5-formylbenzoic acid (see Fig. 3 ), make intersubunit salt-bridge interactions with the guanidinium group of αArg141 on the opposite alpha subunit in the T structure that stabilizes the T state. The left-shifting aromatic aldehydes lack these carboxylate moieties.


With the important lesson learned about potential toxicity issues with covalent AEHs, it was realized that, for any antisickling agent to become a successful drug candidate, clinicians must start with a nontoxic or low-toxicity scaffold in designing new agents. Abraham revisited the food additive vanillin, which had previously been shown by Zaugg and colleagues to have antisickling activity, which he translated into a phase-I clinical trial. Although nontoxic, like valeresol, vanillin was not orally bioavailable, and the phase-I clinical study was terminated. Aldehydes are subject to aldehyde dehydrogenase–mediated oxidative metabolism in human RBCs and liver, which may have been rapid for both vanillin and valeresol, explaining their non–oral bioavailability. A decade later, the prodrug of vanillin, in which the aldehyde group was protected by l -cysteine to form a thiazolidine complex (thiazovanillin; see Fig. 3 ), was shown to have significantly improved oral pharmacokinetic and pharmacodynamic properties compared with the corresponding free aldehyde vanillin, suggesting a viable strategy to improve oral bioavailability, as well as the efficacy of similar antisickling aldehydes.


In a collaborative effort between Don Abraham, Martin Safo, Osheiza Abdulmalik, and Toshio Asakura, 5-HMF (see Fig. 3 ) was shown to have remarkable antisickling activity. A single oral dose of 100 mg/kg of 5-HMF was sufficient to protect transgenic sickle mice from death from acute pulmonary sequestration of sickle cells after a hypoxic challenge, whereas chronic administration of up to 375 mg/kg/d of 5-HMF for 2 years was nontoxic to rats or mice. Acute oral median lethal dose for 50% of the test population (LD 50 ) values for rats were 2.5 to 5.0 g/kg for 5-HMF (US Environmental Protection Agency, 1992). 5-HMF also shows no in vitro cytotoxic effects on RBCs, and plasma proteins do not inhibit its binding to intracellular Hb.


Crystal structures of 5-HMF and similar furfural analogues ( Fig. 4 ) in complex with the quaternary T or R2 structure show that the compounds form Schiff-base interactions with the αVal1 nitrogen in a symmetry-related fashion. The binding of 5-HMF to the R2 structure adds additional intersubunit interaction through a series of direct and intricate water-mediated interactions that tie the two alpha subunits together and restrict the transition to the T state (see Fig. 4 ). The high specificity of 5-HMF for Hb is most likely caused by this intricate and strong hydrogen-bond network. Other less potent antisickling aldehydes, such as vanillin and furfural, lack this intricate sheath of water molecules, explaining their reduced allosteric activity. In contrast with binding to the R2 structure, binding of 5-HMF, as well as other aldehydes, to the wider T structure alpha cleft always results in weak binding of the compounds, which does not seem to add additional intersubunit interaction across the dimer interface. Binding, however, disrupts a T-state water-mediated bridge between α1Val1 and the opposite subunit residue α1Arg141, effectively destabilizing the T structure and shifting the equilibrium to the R state.


Mar 1, 2017 | Posted by in HEMATOLOGY | Comments Off on Therapeutic Strategies to Alter the Oxygen Affinity of Sickle Hemoglobin

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