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

Abstract

Abstract 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. Disclosures Venitz: 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.