Anti-sickling Compounds Research Articles

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 are not available, and will not be available for many years, for the vast majority of the millions of patients suffering from this disease, who live in under-resourced countries. Hydroxyurea is considered standard therapy for patients with SCD and would be even more therapeutic if it increased fetal hemoglobin in all red blood cells, which it does not. What is urgently needed now is an inexpensive oral drug, in addition to hydroxyurea, that inhibits sickle-hemoglobin polymerization in all cells. To this end, we have just completed a phenotypic screen of the 12,657 compounds of the Scripps ReFrame drug repurposing library using our recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from individuals with sickle trait (HbAS), the heterozygous condition. The ReFrame library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials with extensive drug annotation in an open-access format. Compounds, which inhibit sickling in our assay at concentrations that can be achieved in human serum, without side effects that would be deleterious for SCD patients, can be brought to clinical trials with very few or even no pre-clinical studies. From dose-response measurements, we discovered that 106 of the 12,657 compounds are anti-sickling at concentrations ranging from 31 nM to 10 microM. We also showed that compounds that inhibit sickling of trait cells are effective with SCD cells. As many as 21 of the 106 anti-sickling compounds should emerge as drugs. This estimate is based on a comparison of the distribution of inhibitory concentrations in our assay with the known distribution of free concentrations of oral drugs in human serum (see Figure). Moreover, the expected therapeutic effect for each level of inhibition can be predicted from our measurements of sickling times for cells from individuals with sickle syndromes of differing clinical severity. We believe our results represent a breakthrough in drug therapy by serving as a catalyst for us and others to develop one or more of these 106 anti-sickling compounds into effective drugs for treating SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

High-Throughput Assay to Screen Small Molecules for Their Ability to Prevent Sickling of Red Blood Cells.

Sickle cell disease (SCD) is an inherited disorder of hemoglobin (Hb); approximately 300,000 babies are born worldwide with SCD each year. In SCD, fibers of polymerized sickle Hb (HbS) form in red blood cells (RBCs), which cause RBCs to develop their characteristic “sickled” shape, resulting in hemolytic anemia and numerous vascular complications including vaso-occlusive crises. The development of novel antisickling compounds will provide new therapeutic options for patients with SCD. We developed a high-throughput “sickling assay” that is based on an automated high-content imaging system to quantify the effects of hypoxia on the shape and size of RBCs from HbSS SCD patients (SS RBCs). We used this assay to screen thousands of compounds for their ability to inhibit sickling. In the assay, voxelotor (an FDA-approved medication used to treat SCD) prevented sickling with a z′-factor > 0.4, suggesting that the assay is capable of identifying compounds that inhibit sickling. We screened the Broad Repurposing Library of 5393 compounds for their ability to prevent sickling in 4% oxygen/96% nitrogen. We identified two compounds, SNS-314 mesylate and voxelotor itself, that successfully prevented sickling. SNS-314 mesylate prevented sickling in the absence of oxygen, while voxelotor did not, suggesting that SNS-314 mesylate acts by a mechanism that is different from that of voxelotor. The sickling assay described in this study will permit the identification of additional, novel antisickling compounds, which will potentially expand the therapeutic options for SCD.

Characterization of a Genetically Engineered HUDEP2 Cell Line Harboring a Sickle Cell Disease Mutation As a Potential Research Tool for Preclinical Sickle Cell Disease Drug Discovery

Sickle cell disease (SCD) is a genetic disorder caused by a missense mutation in the beta-globin gene [Glu6Val] resulting in the formation of HbS (sickle hemoglobin). HbS polymerizes under low oxygen tension causing an array of severe pathophysiological complications, including vaso-occlusion, pain and hemolytic anemia. Traditionally, research efforts toward studying anti-sickling effects of various pathways and compounds in vitro have been made using sickle cell patient derived samples. However, such studies are limited based on sample availability and a large degree of phenotypic variability. Thus, a cultured cell system with a sickle mutation mimicking human sickle cell erythroid cells is a potentially useful tool that can be used to interrogate anti-sickling compound action and to study various facets of sickle cell phenotype. In this study, we generated a genetically engineered sickle mutation (GAG to GTG) in HUDEP2 cell line using CRISPR Cas9 gene editing and homology directed repair mechanism. HUDEP2 cells are immortalized human erythroid progenitor cells, obtained from the Riken Institute and were cultured as described (Kurita et al 2013). CRISPR Cas9 RNP complex with modified guide RNA along with ssODN were electroporated in HUDEP2 cells. Cells were also treated with SCR7, a NHEJ inhibitor to facilitate homology- directed repair events. Cells were sorted for clonal selection, cultured and further screened for homozygous knock-in mutations using digital droplet PCR. The mutation was further confirmed by sanger sequencing. 12 clones consisting of homozygous mutation were identified, with 6 of these clones selected for further characterization. Cells were differentiated for 7 days in culture and hemolysates were analyzed using HPLC. Heterozygote clone showed 32.3% HbS which was comparable to sickle cell trait patient derived blood samples whereas homozygous clones (ssHUDEP2) showed between 71-85% HbS. These results were further confirmed for the presence of HbS using mass spectrometry. Mutant HUDEP2 cell growth and differentiation were similar to the wild-type HUDEP2 cells. To evaluate sickling in these cells we optimized the differentiation protocol to improve the maturation of these cells by withdrawing SCF and doxycycline gradually. Hemoglobin polymerization and resulting shape change in sickle HUDEP cells was determined by subjecting mature ssHUDEP2 cells to hypoxia (1% oxygen) for 3 hours. Upon subjecting to hypoxia, ssHUDEP2 cells showed morphological changes indicative of HbS polymerization. These morphological changes were absent in wild type cells under similar conditions of hypoxia. In addition, ssHUDEP2 cells pre-treated with 25µM GBT440, a known anti-sickling compound, for 1 hour prior to hypoxia incubation elicited a reduction in the shape change associated with hypoxia, suggesting inhibition of hemoglobin polymerization and sickling. These results suggest that genetically-engineered sickle mutant HUDEP cells represent a preclinical model for development of anti-sickling compounds for the potential treatment of Sickle Cell Disease and to explore novel mechanistic pathways. Disclosures Gupta: Sanofi: Employment. Sturtevant:Sanofi: Employment. Vieira:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. Demers:Sanofi: Employment.

LC-MS Analysis of Anti-Sickling Compounds in Cord Blood Derived RBCs Demonstrates Modification of Fetal Hemoglobin and Globin Chain Binding Preferences

Sickle cell disease (SCD) is a genetic hemoglobinopathy driven largely by a single codon mutation of the β-globin gene resulting in polymerization of hemoglobin S (HbS). Anti-sickling approaches that involve increasing the oxygen affinity of HbS to treat SCD are under development and offer the potential to directly prevent HbS polymerization and its downstream pathophysiology. Two such compounds, 5-hydroxymethylfurfural (5HMF) and voxelotor (GBT440) have entered clinical trials for SCD with promising results and exert their therapeutic effects by modifying the N-terminus of HbS α-globin chains to form a reversible Schiff base. Formation of this N-terminal adduct stabilizes the oxygen-bound R-state (in the R2 conformation) that increases the oxygen affinity of the altered HbS and delaying the polymerization of HbS. In addition, genetic and small molecule therapies designed to increase fetal hemoglobin (HbF) expression hold great potential for the treatment of SCD. Increasing the percentage of HbF in RBCs significantly slows sickling kinetics without affecting oxygen delivery. Combination approaches of high-O2-Hb modification with HbF inducing therapies clinically could result in increased efficacy in the treatment of SCD, but the impact of hemoglobin modifiers on fetal hemoglobin has not been reported. Our present studies investigated the effects of 5HMF and voxelotor in HbF-rich umbilical cord blood derived RBCs.HbF-rich (60-90%) RBCs were isolated from cord blood and incubated with commercially available 5HMF and voxelotor synthesized in-house. The effect of these compounds on hemoglobin oxygen affinity was determined by measuring the p50 of the oxygen saturation curve in whole cells. Sites of modification were determined directly by incubating compounds with the purified RBC lysate, stabilizing the N-terminal adduct by reduction to the amine, and analysis of the resulting modification by LC-MS. Similar to the reported p50 shifts with normal adult hemoglobin (HbA) and HbS, 5HMF and voxelotor increased the oxygen-binding affinity of HbF with an EC50 of 7.9 mM and 560 mM respectively. 1 mM voxelotor lowered cord RBC p50 to 4 mmHg in vitro. LC-MS analysis showed that 5HMF exclusively modified the N-terminus of the α-globin chain, with no modification of b-globin and g-globin chains. Unexpectedly, the α-globin, β-globin and γ-globin chains were all modified by voxelotor following incubation with cord blood. Voxelotor was also shown to modify both α-globin and the β-globin or βS-globin chains on purified HbA or HbS, respectively. These data contrast with published crystallography data demonstrating that voxelotor selectively modifies a single α-globin chain in CO-ligated HbS (Oksenberg et al 2016).Although anti-sickling aromatic aldehydes have similar effects on the oxygen binding affinity of HbA, HbS and HbF, they can vary in their selectivity for modification of the α-globin and beta-like chains of HbF, HbA, and HbS (Abraham et al. 1995). To further investigate our data with voxelotor and increase our understanding of this class of molecules, other hemoglobin modifying aldehyde molecules such as 5-formylsalicyclic acid (5FSA), tucaresol and velaresol (BW12C) will be examined. DisclosuresVieira:Bioverativ a Sanofi Company: Employment. Hong:Bioverativ a Sanofi Company: Employment, Equity Ownership. Desai:Bioverativ a Sanofi Company: Employment, Equity Ownership. Safo:Bioverativ a Sanofi Company: Consultancy; Virginia Commonwealth University: Employment. Light:Bioverativ a Sanofi Company: Employment, Equity Ownership.

Sickle Human Umbilical Cord Derived Erythroid Progenitor Cells (S-HUDEP2): An Ideal in-Vitro System for Screening Anti-Sickling Compounds for Sickle Cell Disease

Background: Sickle Cell Disease (SCD) is a devastating inherited disease, characterized by polymerization of sickle hemoglobin under deoxygenated conditions that can lead to acute pain crises, ischemia, and chronic organ damage. Pharmacologic anti-sickling agents that decrease polymerization are currently under investigation, however there is no consistent in vitro system to study these compounds; whole patient blood, subject to clinical variability and limited supply, is most often used. Human Umbilical Cord Derived Erythroid Progenitor 2 cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell (HSC) derived erythroid precursor cell line that can differentiate into red blood cells. We have engineered S-HUDEP2 cells to express sickle hemoglobin (HbS) via CRISPR/Cas9 gene editing. We hypothesized that this cell line will sickle under hypoxic conditions, produce dense red blood cells (DRBC, red cells with a density>1.11 mg/mL that are dehydrated and prone to sickling. If these intrinsic, essential SCD RBC properties are found, we propose to use this novel cell line to screen drug compounds for anti-sickling capabilities.Methods: S-HUDEP cells were cultured as previously described (Kurita et al, 2013). %HbS and %HbA produced by parent HUDEP and S-HUDEP2 cells were measured by high performance liquid chromatography (HPLC). Hypoxia was induced by placing the cells at 2% O2 for four hours. Parent HUDEP and S-HUDEP2 cells were then fixed with glutaraldehyde and Giemsa stained. % sickling estimated at 40x magnification by a pathologist blinded to cell group counting sickle forms out of 1000 cells. The percentage of dense red blood cells (DRBCs) was quantified by an ADVIA hematology analyzer (Siemens). S-HUDEP2 cells were dosed with 0, 2.5 and 5 µM 5-hydroxymethylfurfural (5-HMF) and 75, 150, and 300 µM GBT440, two known anti-sickling agents, on day ten and day 14 of culture for one hour, subjected to hypoxic conditions and % sickling quantified as described above.Results: S-HUDEP2 cells express 98% HbS. Under hypoxia, 20% of S-HUDEPs sickle at day 10 of differentiation; 30% of S-HUDEP2 cells sickle at Day +14 of differentiation. Parent HUDEP-2 cells, which produce 98% HbA, did not sickle under hypoxic conditions at any stage of differentiation. 70% of S-HUDEP2 cells were determined to be DRBC under hypoxia at Day 10 and 14 time points; parent HUDEP-2 cells did not produce DRBC under hypoxia. Treatment of S-HUDEP2 cells with 5μM of 5-HMF and 150 μM of GBT440 reduced sickling by 40-50% under hypoxic conditions compared with untreated S-HUDEP2 cells (p<0.01), and reduced %DRBC by 30% (p<0.01).Conclusions: S-HUDEP2 cells express HbS, form DRBC, and sickle under hypoxic conditions, just like erythroid precursors and mature red blood cells from individuals with SCD. Exposure to two anti-sickling agents, one of which is currently in Phase III clinical trials, significantly decreased S-HUDEP2 sickling under hypoxic conditions, and reduced %DRBC, a marker of disease severity. Advantages of S-HUDEPs over patient samples include genetic and phenotypic uniformity, no human pathogens, and availability to groups without access to patient samples. We therefore conclude that S-HUDEP2s have utility in clinical research, may be used to screen anti-sickling and anti-dense cell compounds in vitro, and may lead to identification of new therapeutic options for SCD patients. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Some Salicylaldehyde-derived Baylis-Hillman Adducts and Coumarin Derivatives as Potential Antisickling Compounds

Abstract Some salicylaldehyde-derived Baylis-Hillman adducts and 3-(chloromethyl)coumarins have been synthesised and evaluated in vitro for their antisickling activities. The compounds were screened for inhibitory and reversal activity against mutated haemoglobin (HbSS) in red blood cells at four different concentrations (4 mg/mL, 2 mg/mL, 1 mg/mL and 0.5 mg/mL) as a measure of their antisickling potentials. Among the synthesized compounds, 6-chloro-3-(chloromethyl)coumarin 4d showed the highest inhibitory activity (83.75±1.90%), followed by 6-chlorocoumarin-3-methylsulfinic acid 5d (80.90 ±0.91%) and the least was tert-butyl-3-hydroxy- 3-(2-hydroxyphenyl)-2-methylenepropanoate 3a (33.33±1.86%). The results obtained from the reversal antisickling experiment showed that the percentage of sickle cells able to revert to the normal biconcave shape was dose dependent. Compound 5d had the highest reversal activity (66.49±1.39%) followed by 6-bromo-3- (chloromethyl)coumarin 4c (59.66±2.95) and 4d (55.50±1.95%) at 4 mg/mL. Compound 4c had higher reversal activity than the standard p-hydroxybenzoic acid at 2 mg/mL, 1 mg/mL and at 0.5 mg/mL. The 3-substituted coumarins 4a-d, and 5d had higher inhibitory antisickling activities than their Baylis-Hillman precursors 3a-d. Effect of 4a-d and 5d on the rate of polymerization of sickle cell heamoglobin was further studied spectrophotomerically using hemolysate of HbSS. The considerable inhibitory and reversal activities of these compounds make them good candidates for further antisickling studies.

Nanomolar anti-sickling compounds identified by ligand-based pharmacophore approach

Adenosine receptors are considered as potential targets for drug development against several diseases. The discovery of subtype 2B adenosine receptors role in erythrocyte sickling process proved its importance to neglected diseases such as sickle cell anemia, which affects approximately 29.000 people around the world, but whose treatment is restricted to just one FDA approved drug (hydroxyurea). In order to widen the therapeutic arsenal available to treat sickle cell anemia patients, it is imperative to identify new lead compounds that modify the sickling course and not just its symptoms. In order to accomplish this goal, ligand-based pharmacophore models that differentiate true ligands from decoys and enlighten the structure-activity relationship of known RA2B antagonists were employed screen the lead-like subset of the ZINC database. Following a chemical diversity analysis, 18 compounds were selected for biological evaluation. Among them, one molecule Z1139491704 (pEC50 = 7.77 ± 0.17) has shown better anti-sickling activity than MRS1754 (pEC50 = 7.63 ± 0.12), a commercial RA2B antagonist. Moreover, these compounds exhibited no cytotoxic effect at low micromolar range on mammalian cells. In conclusion, the sound development of validated ligand-based pharmacophore models proved essential to identify novel chemical scaffolds that might be useful to develop anti-sickling drugs.

Rheological effect of activation of the KCl-cotransport pathway in normal and sickle erythrocytes

Activation of the KCl-cotransport pathway in vivo is a potentially important cause of dehydration and rheological impairment of sickle cells. Its activation in vitro is a useful new method for investigating the rheological effects of putative anti-sickling compounds, including specific inhibitors of the KCl-cotransporter

GTx011, an Anti-Sickling Compound, Improves SS Blood Rheology By Reduction of HbS polymerization Via Allosteric Modulation of O2 Affinity

Sickle cell disease is caused by a mutation in the β-globin gene leading to production of hemoglobin S (HbS). HbS polymerizes under hypoxic conditions, leading to changes in cytoplasmic viscosity and formation of rigid, non-deformable sickled red blood cells (RBC). Loss of RBC deformability leads to abnormal blood rheology, diminished oxygen (O2) delivery and contributes to vaso-occlusion. GTx011 is a novel orally bioavailable small molecule that has been shown to inhibit HbS polymerization in vitro by maintaining oxygenated HbS under hypoxic conditions. We investigated the mechanism by which GTx011 modulates O2 affinity. Although it binds to the N-terminal α chain of Hb via a reversible Schiff base, 1D 1H NMR studies indicated that the compound allosterically influenced the intra-dimer interface of Hb (α122His and α103His), and the distal valine surrounding heme pockets for both the α and β chains, suggesting a unique solution phase structure. GTx011’s unique allosteric action upon the heme pocket allows it to improve O2 affinity without sterically blocking the release of O2. This modulation of O2 affinity delays the onset of HbS polymers and therefore we hypothesized will improve the abnormal blood rheology observed in SS blood.We report here the beneficial effect of GTx011 on SSRBC deformability using four independent whole cell systems: 1) the profile of SSRBC during passage through a gel filtration column, 2) the ability and corresponding pressure required for SSRBCs to pass through a polycarbonate filter under hypoxic conditions, 3) the tension required to aspirate SSRBCs into a micropipette under low O2 and 4) changes to blood viscosity during deoxygenation. Under deoxygenating conditions, GTx011 dose dependently enabled RBC deformability required for migration of SSRBCs through a gel filtration column. The pressure required to pass SSRBCs through a polycarbonate filter and the tension required to aspirate SSRBCs into a micropipette under hypoxic conditions were also reduced (Table 1). We have established a novel blood viscosity assay, which employs an enzymatic reaction (ascorbic acid/ascorbic oxidase) for deoxygenation of blood, followed by traditional measurements in cone plate viscometers. This protocol enabled us to reduce O2 levels without the technical limitations of using sodium dithionite, laser induced deoxygenation or passive deoxygenation via N2. Time dependent measures of viscosity, at a fixed shear rate, allowed us to quantify GTx011-induced delay in the onset of polymerization, and the increase in cytoplasmic viscosity. As in polymerization experiments, GTx011 dose dependently delayed the onset of hyperviscosity of SS blood under deoxygenating conditions. HbS modification ranging from 10-30%, was sufficient to achieve an improvement in SS blood hyperviscosity (Table 2)These data suggest that GTx011 is a promising agent that has the potential to improve SSRBC deformability and blood viscosity by inhibiting HbS polymerization, the pathophysiologic hallmark of sickle cell disease.Table 1AssayMigrationFilterabilityDeformabilityunit(% deformable cells)(mm Hg)(mDynes/cm2)[HbS]1 mM1 mM1 mM[GTx011](mM)00.5100.5100.51Readout1642893581621291013225Table 2AssayPolymerizationViscosity[HbS]50 μM1.5 mMGTx011 modified HbS(%)0103001030Delay time(min)131833212631 DisclosuresPatel:Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Consultancy. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Metcalf:Global Blood Therapeutics: Consultancy, Equity Ownership. Sinha:Global Blood Therapeutics: Employment, Equity Ownership.

Liganded Hb ζ2βS2 Exhibits Antipolymer Activity Despite a T-like Quaternary Structure

Important therapeutic approaches to sickle-cell disease (SCD) are based upon the observation that the abnormal properties of Hb S (Hb α2βS2) can be mitigated by exchanging the pathological βS-globin subunit for a related β-like subunit. We previously demonstrated that exchange of the non-pathological α-globin subunit for a ζ-globin subunit (a developmentally silenced globin that can be derepressed both by natural and experimental conditions) inhibits deoxyHb S polymer assembly in vitro and reverses hematological, biochemical, and physiological characteristics of SCD in mouse models in vivo. While its therapeutic potential is clear, the underlying structural basis for the profound antipolymer activity of ζ-substituted Hb S (Hb ζ2βS2) is less certain. X-ray crystallographic studies conducted at 1.95Å resolution revealed that liganded (CO-) Hb ζ2βS2 is trapped in a tense (T-state) quaternary structure, rather than in a relaxed (R-state) structure that is characteristic of nearly all liganded hemoglobins. Specifically, CO-Hb ζ2βS2 exhibited several intact T-state intersubunit salt-bridge/hydrogen-bond interactions, a preserved T-state ζ1-β2 (ζ2-β1) interface, and a characteristically enlarged T-state central water cavity and β-cleft. This structure wrongly predicts that liganded Hb ζ2βS2 will be included, rather than excluded, from the deoxyHb S polymer; and suggests that changes in the positions or the biochemical identities of individual amino acids, rather than the overall quaternary structure of liganded Hb ζ2βS2, are the chief determinants of its antipolymer activity.To define key differences in the structures of T-state deoxyHb α2βS2 and CO-Hb ζ2βS2, we superposed their corresponding globin subunits and calculated the specific displacement of individual amino-acid residues as root mean square deviation (rmsd) values. Among βS-chain residues, α→ζ exchange effects a significant 1.9Å shift in the position of the pathological βSVal6, and correspondingly large displacements of βThr4 (2.2Å) and βAsn19 (1.4Å); each repositioning predicts weakening of an intermolecular interaction that would otherwise stabilize the deoxyHb S polymer. Similar superposition analyses of the α and ζ chains reveal a significant displacement of αPro114 (1.3Å), a well-described determinant of deoxyHb S polymerization that is conserved between the two α-like subunits. Three additional α-chain residues that stabilize the deoxyHb S polymer undergo nonconservative replacement in the ζ-globin chain, but are not materially repositioned: αHis20→ζGln (basic→neutral polar), αAsn68→ζAsp (neutral polar→acidic), and αAsn78→ζGly (neutral polar→neutral). While all three replacements are predicted to weaken or ablate intermolecular contacts, the αHis20→ζGln substitution is particularly noteworthy as it reproduces the specific mutation that defines the naturally occurring anti-sickling variant αLe Lamentin.Finally, we considered the possibility that ζ-substituted Hb S is fully excluded from the deoxyHb S polymer–and therefore reduces the rate of its assembly–by comparing the crystal packing of the two hemoglobins. While deoxyHb α2βS2 packs in a familiar two-strand structure, CO-Hb ζ2βS2 assembles into a unique trimeric arrangement comprising three lateral heterotetramers, each of which interacts with an axial heterotetramer that is constituent to a separate trimer assembly. This remarkable structure is sustained by intermolecular interactions that are distinct from those observed for deoxyHb S. Moreover, the calculated buried solvent-accessible surface area for CO-Hb ζ2βS2 (4806Å2) is nearly two-fold higher than for deoxyHb α2βS2 (2510Å2), suggesting that Hb ζ2βS2 exists in solution as a stable trimer of heterotetramers, and validating the hypothesis that Hbs α2βS2 and ζ2βS2 do not co-assemble in solution.In sum, our crystal analyses indicate that the antipolymer activities of liganded Hb ζ2βS2 arise through movements in the positions of βS-chain residues, and through changes in the identities of α-chain residues. Our studies also demonstrate a novel packing structure for T-state liganded Hb ζ2βS2 that is consistent with its exclusion from the deoxyHb S polymer. These data account for the significant antipolymer activity of ζ-substituted Hb S, and recommend the utility of therapeutic approaches to SCD that are based upon α-globin subunit exchange. DisclosuresSafo:Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.

Novel Structurally-Modified Allosteric Effectors of Hemoglobin Exhibit Superior Antisickling Properties

Sickle cell disease (SCD) continues to cause significant morbidity, mortality and healthcare disparities. Despite considerable progress in understanding the underlying pathophysiology and investigating various therapeutic strategies, novel pharmacologic approaches to ameliorate SCD continue to hold immense potential and promise, especially for patients in developing countries.Our group and others have recently renewed and refocused attention to candidate drugs that directly bind to hemoglobin (Hb) and increase oxygen (O2) affinity, preventing the fundamental pathophysiology of the disease, i.e., sickle Hb (Hb S) polymerization and red blood cell (RBC) sickling. While several candidate drugs have shown biological activity in-vitro, ex-vivo and in animal studies, their ultimate success in clinical studies was hampered by toxicity concerns and/or low oral bioavailability. Recent promising reports from a phase I/II study on 5-HMF renews optimism for this therapeutic approach.We reasoned that modifications of vanillin–a previously reported antisickling agent and food constituent without known toxicities–to enhance its efficacy, would represent a feasible approach in rationally developing clinically useful candidate drugs. Consequently, we designed and synthesized two classes of compounds: INN and TD series. The former are pyridyl derivatives of vanillin, rationalized to stereospecifically inhibit deoxy-Hb S polymer formation while increasing the fraction of the soluble oxy-Hb S in regions of low O2 tension. The TD compounds represent further modification of corresponding INN compounds (with a methoxyl group on the pyridine ring), rationalized to exhibit similar dual antisickling effects, but with enhanced direct polymer destabilization properties.We subjected a prototypical compound from each class (INN-270 and TD-7) to our battery of exploratory in-vitro assays, specifically: 1) rates of Hb S binding/modification, 2) corresponding change in O2 affinity, 3) direct inhibition of Hb S polymerization, and 4) inhibition of RBC sickling under hypoxia. We incubated 0.5, 1, or 2 mM of either INN-270 or TD-7 with RBCs from patients with homozygous SCD, under hypoxia (4% O2/96% N2 gas mixture) in a shaker-incubator at 37 ˚C for 3 h. Assays were conducted in at least three replicates utilizing different samples on different days. At the conclusion of each assay, aliquot samples (~ 10 μl each) were drawn into a fixing solution under hypoxia to preserve RBC morphology for analyses. Residual RBC suspensions were washed, hemolyzed, and subjected to: cation-exchange HPLC (to determine Hb modification); P50 analyses to establish change in O2 affinity; and temperature-dependent delay time studies to establish a delay in Hb S polymerization.Our results show that both compounds permeated RBC membranes without causing hemolysis, bound to and modified intracellular Hb at high levels in a dose dependent manner, increased O2 affinity significantly, and inhibited sickling of RBCs under hypoxia. TD-7 modified Hb S in a dose-dependent manner (to 92.3 ± 5.2 %, n=4 at 2 mM), shifted O2 equilibrium to the left (Δp50 = 45.6 ± 8.2 %, n=3 at 2 mM), and inhibited RBC sickling (by 95 -100 %, n=4). Preliminary delay time analyses also showed that at 2 mM, TD-7 increased the Hb S polymerization times from 18.1 ± 1.0 min to 24.5 ± 0.5 min. INN-270 showed a similar profile, however with a lower efficacy (at 2 mM) for Hb S modification (to ~ 75 %), Δp50 of 40.3 %, sickling inhibition by ~ 70 %, and increased delay times from 15.6 ± 0.5 min to 19.7 ± 1.0 min.We have elucidated the dual antisickling mechanism of action of INN-270 and TD-7 by X-ray crystallography. Two molecules of each compound bind to Hb via Schiff-base, and a series of hydrogen-bond/hydrophobic interactions that favor a high-O2-affinity Hb state. Importantly, the methoxyl group on the pyridine ring of TD-7 forms hydrogen-bond interactions with the surface-located αF-helix, resulting in a conformational change, possibly explaining the improved potency.Based on our results, both TD7 and INN 270 exhibited greater than a 40- and 3-fold superiority in efficacy compared to vanillin and 5-HMF, respectively. We conclude that our findings justify a prospective, structure-based approach to designing novel antisickling agents with enhanced potency. In-vitro/ex-vivo murine and human PK/PD studies are currently ongoing to help guide planned in-vivo PK/PD studies in mice. DisclosuresVenitz:Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: Consultancy. Safo:Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.

New isocratic high-performance liquid chromatographic procedure to assay the anti-sickling compound hydroxyurea in plasma with ultraviolet detection

A new procedure using high-performance liquid chromatography (HPLC) with ultraviolet detection to assay hydroxyurea (HU) levels in plasma has been developed. The drug was isolated from plasma by a direct deproteinization process with sulfosalicylic acid. Following neutralization of the acidic supernatant, an aliquot was loaded onto an Aminex HPX-72S column (300×7.8 mm). Chromatography was performed at 55°C using a mobile phase consisting of acetonitrile–0.025 M ammonium sulfate buffer (pH 8.5) including 0.1% triethylamine, 0.01 M sodium sulfate, and 5 m M sodium heptane sulfonate. The UV absorbance of effluent was monitored at 214 nm. A flow-rate of 0.8 ml/min was used for analyzing HU in both human and mouse plasma. Under these conditions, the drug eluted at 12.6 min. The assay possessed linearity up to 425 μg/ml, with a lower limit of quantitation of 3.32±0.0004 μg/ml (mean±S.D., n=10). Intra-day and inter-day coefficients of variation were less than 8.5% and 8.7% respectively. Absolute differences were less than 7.4%. The method has been employed in clinical studies and the sensitivity of the assay was shown to be adequate for characterizing the plasma pharmacokinetics of HU in mice. In conclusion, the procedure described herein could be ideally suited for therapeutic monitoring of hydroxyurea.

Additive in vitro effects of anti-sickling drugs.

To study the effect of anti-sickling drugs on cellular dehydration induced by entry of Ca, sickle cells were subjected to cyclical oxygenation-deoxygenation for 15 h in Ca-containing buffer. The consequential loss of cation (K) via the Ca-dependent K efflux (Gardos) channel caused cell dehydration and loss of deformability. Inhibition of a specific fraction of Ca entry by verapamil had no rheologically protective effect, whereas inhibition of the Gardos channel by clotrimazole or nitrendipine had a marked protective effect. When Gardos channel inhibition (by either clotrimazole or nitrendipine) was combined with stabilization of the oxy-conformation of sickle haemoglobin (by the substituted benzaldehyde 12C79), an additive protective rheological effect was achieved with 60-78% reduction in clogging rate of 5 microns diameter pores when compared with no drug. Therapeutic use of anti-sickling compounds in combination may achieve increased efficacy with lower toxicity.

Pharmacological modification of oxygen affinity improves deformability of deoxygenated sickle erythrocytes: a possible therapeutic approach to sickle cell disease.

1. The formation of polymerized haemoglobin S in sickle cells is critically dependent on the concentration of deoxygenated haemoglobin so that compounds which increase the oxygen affinity of haemoglobin S are potential anti-sickling agents. 2. BW12C [5-(2-formyl-3-hydroxyphenoxy)pentanoic acid] and BWA589C [4-(2-formyl-3-hydroxyphenoxymethyl)benzoic acid] are aromatic benzaldehydes that cause a dose-dependent left-shift of the oxygen saturation curve of haemoglobin S by stabilization of its oxy-(R)-conformation. 3. A 5 micron pore filtration method, which is highly sensitive to polymerization of haemoglobin S, was used to demonstrate a significant improvement in the deformability of deoxygenated sickle erythrocytes at concentrations (0.75-1.5 mmol/l] of BW12C and BWA589C that are achievable in vivo. Both compounds may therefore be of value for the treatment of sickle cell disease. 4. Filtration of sickle cells through pores of 5 microns diameter is a sensitive technique for evaluating the rheological effects of potential anti-sickling compounds.

Irreversible erythrocyte volume expansion induced by tellurite.

Tellurite (K2TeO3) has been suggested as a potential anti-sickling compound because it causes a selective increase in the water content of RBC. To investigate the conditions underlying the increase in RBC volume due to tellurite, normal RBCs were incubated with the compound in a physiological medium and the cells washed with a 10-fold volume of the medium. The washed cells were then incubated at 24 degrees C for periods up to 4 h and the following parameters were determined: MCV, MCH, MCHC and supernatant haemoglobin concentration by standard methods, the density distribution profile by phthalate esters and cell morphology by scanning electron microscopy (SEM). The effect of hypertonic PBS on the tellurite-treated cells was also tested. K2TeO3 induced concentration and time dependent increases in MCV and decreases in MCHC without any apparent change in MCH. The median density and the transitional 60% density range of the cell distribution profile respectively decreased and increased in proportion to [K2TeO3] and time. Hypertonic PBS did not inhibit or reverse the tellurite-induced changes in MCV and MCHC. SEM and photovolumetric measurements demonstrated tellurite-induced large vesicles ranging in size from 24 to 32 micron 3. The proportion of these vesicles increased with time and K2TeO3 concentration. Since tellurite is an oxidant, these findings suggest that its influx into the red cell results in irreversible reactions that disrupt the ion and water regulatory properties of the membrane.

Reduction in potency and reversal of left-shifting activity of BW12C with the major and minor components of chicken hemoglobin

The effects of the left-shifting, anti-sickling compound BW12C (5-(2-formyl-3-hydroxyphenoxy)pentanoic acid) on the oxygen saturation curve of whole chicken blood and the isolated major (AII) and minor (AI) components of chicken hemoglobin have been studied. The results support the postulated major binding mode for BW12C to human hemoglobin of bridging between the α-chain terminal amino groups in the oxy conformation with an important hydrophobic component contributed mainly by Pro 77α residues. In chicken AH (Pro 77 α → Ser) BW12C still left-shifts at high concentrations but its potency is greatly reduced (at least 10-fold). In chicken AI (Pro 77 α → Ser and Val 1 α → Met) BW12C is a right-shifter with a potency comparable to that of 2,3-diphosphoglycerate suggesting that binding at the β-chain termini in the deoxy conformation is now dominant with α-chain binding no longer significant.

Characterization of the binding of the anti-sickling compound, BW12C, to haemoglobin.

The anti-sickling agent BW12C [Beddell, Goodford, Kneen, White, Wilkinson & Wootton (1984) Br. J. Pharmacol. 82, 397-407] was designed to left-shift the oxygen saturation curve of haemoglobin (HbA) by preferential binding to the oxy conformation at a single site between the terminal amino groups of the alpha-chains through Schiff's base formation, ionic and hydrophobic interactions. In the present work, Schiff's base linkages formed with [14C]BW12C were reduced with NaBH4 and the alpha- and beta-globin chains separated. Under oxy conditions at a molar ratio of 2:1, the covalently bound BW12C is localized almost exclusively on a single alpha-chain; tryptic digestion confirms the terminal amino group (alpha 1-valine) as the reaction site, in accord with the design hypothesis. However, about half the labelled BW12C is released on tetramer disruption, suggesting the presence of additional non-covalent binding. Under deoxy conditions, alpha- and beta-chains are labelled approximately equally, and at higher molar ratios additional binding in both oxy and deoxy conditions is seen. Isoelectric-focusing studies under oxy conditions show a complex pattern of modified bands for both HbA and HbA1c (blocked beta-terminal amino groups) but no modification for HbA carbamylated at both alpha- and beta-terminal amino groups or at the alpha-chains only, again confirming the alpha-terminal amino region as the main interaction site. Equilibrium dialysis measurements under oxy conditions indicate two strong binding sites with a binding constant of less than 10(-6) M and a number of weaker binding sites. The present data thus confirm that BW12C binds at the intended locus but reveal additional non-covalent binding at an undefined site, and weaker binding through Schiff's base formation with other amino groups.

Evaluation of covalent antisickling compounds by PO2 scan ektacytometry

The ektacytometer, a device to measure erythrocyte flexibility, has been used to evaluate antisickling agents that covalently modify hemoglobin S (HbS). The instrument has been adapted to produce a continuous gradient of oxygen pressure in the measuring cuvette, which permitted the rapid determination of sickle cell rigidity over the complete oxygenation range. Inspection of curves allows classification of the compounds according to their mode of action: altering oxygen affinity or increasing deoxy-HbS solubility. Reagents that modify amino groups, thiols, and histidine, as well as a crosslinking agent, were examined. The method directly evaluates deoxygenated cell deformability rather than cell shape. Many of the compounds that are effective in preventing the morphological sickling of deoxygenated sickle cells do not necessarily restore cell deformability. The method also readily detects membrane damage brought about by covalent agents that nonspecifically derivatize membrane proteins. Cystamine and pyridoxal appear to improve deformability in deoxygenated SS cells at concentrations that do not damage the membrane. This method, which examines the intact cell, fills a gap in the available experimental techniques for drug evaluation, between studies of isolated hemoglobin and in vivo studies.

Evaluation of Covalent Antisickling Compounds by Po2 Scan Ektacytometry

The ektacytometer, a device to measure erythrocyte flexibility, has been used to evaluate antisickling agents that covalently modify hemoglobin S (HbS). The instrument has been adapted to produce a continuous gradient of oxygen pressure in the measuring cuvette, which permitted the rapid determination of sickle cell rigidity over the complete oxygenation range. Inspection of curves allows classification of the compounds according to their mode of action: altering oxygen affinity or increasing deoxy-HbS solubility. Reagents that modify amino groups, thiols, and histidine, as well as a crosslinking agent, were examined. The method directly evaluates deoxygenated cell deformability rather than cell shape. Many of the compounds that are effective in preventing the morphological sickling of deoxygenated sickle cells do not necessarily restore cell deformability. The method also readily detects membrane damage brought about by covalent agents that nonspecifically derivatize membrane proteins. Cystamine and pyridoxal appear to improve deformability in deoxygenated SS cells at concentrations that do not damage the membrane. This method, which examines the intact cell, fills a gap in the available experimental techniques for drug evaluation, between studies of isolated hemoglobin and in vivo studies.

The effects in volunteers of BW12C, a compound designed to left-shift the blood-oxygen saturation curve.

BW12C, a potent left-shifting anti-sickling compound in vitro, was administered to normal healthy male Caucasian volunteers. Doses of 2-20 mg kg-1 given by intravenous infusion over 1 h caused a dose-dependent left-shift of the blood-oxygen saturation curve and at the highest dose some 16% of the haemoglobin existed in a high affinity form. Peak left-shift was observed at the end of infusion and decayed thereafter with a mean half-life of approximately 3 h. There were no adverse systemic effects, either clinical, biochemical or haematological, but there was some local irritation at the intravenous infusion site if the infusion was too concentrated. Pharmacokinetic measurements indicated uptake into erythrocytes, low levels in plasma and a volume of distribution not appreciably greater than the blood volume. A pilot radiolabel study indicated extensive metabolism with elimination into the urine.