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. 2022 May 12;12(5):696.
doi: 10.3390/biom12050696.

Design, Synthesis, and Antisickling Investigation of a Nitric Oxide-Releasing Prodrug of 5HMF for the Treatment of Sickle Cell Disease

Rana T Alhashimi  1 , Mohini S Ghatge  1 , Akua K Donkor  1 , Tanvi M Deshpande  2 , Nancy Anabaraonye  3 , Dina Alramadhani  1 , Richmond Danso-Danquah  1 , Boshi Huang  1 , Yan Zhang  1 , Faik N Musayev  1 , Osheiza Abdulmalik  3 , Martin K Safo  1
Affiliations

Design, Synthesis, and Antisickling Investigation of a Nitric Oxide-Releasing Prodrug of 5HMF for the Treatment of Sickle Cell Disease

Rana T Alhashimi et al. Biomolecules. .

Abstract

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies.

Keywords: antisickling; hemoglobin; nitric oxide; oxygen equilibrium curve; sickle cell disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of 5HMF and its NO-releasing derivative.
Scheme 1
Scheme 1
5HMF-NO. (a) LiNO3, TFAA, Na2CO3, anhydrous CH3CN, 0 °C, 7 h.
Figure 2
Figure 2
Time-dependent NO release from 5HMF-NO. NO release (in the form of nitrite) by 5HMF-NO (red), 5HMF (cyan), and no test compound (grey). The results are the mean values ± SD for biological duplicate or triplicate experiments. All compounds were solubilized in 2% DMSO. Control experiment without test compound also included 2% DMSO. Final test compound concentration was 1 mM.
Figure 3
Figure 3
Representative figures of Hb modification and OEC shift by 5HMF-NO in sickle blood: (A) Cation exchange HPLC chromatograms showing a dose-dependent Hb modification; (B) OEC curves demonstrating a doses-dependent left-shift in oxygen affinity study.
Figure 4
Figure 4
Functional/biological effects of 5HMF-NO in sickle blood. Hb modification, oxygen equilibrium and antisickling studies were conducted with SS blood suspensions (hematocrit of 20%) incubated with 1, 2 and 5 mM of 5HMF or 5HMF-NO. The final concentration of DMSO was <2% in all samples, including in control samples. The results are the mean values ± SD for two or three biological replicate experiments for the Hb modification and antisickling studies, and single measurement for the OEC study. The differences between the 5HMF and the 5HMF-NO groups were not statistically significant (p values of 0.2, 0.66, and 0.98, respectively). (A) Concentration-dependent modification of Hb S; (B) Concentration-dependent P50 shift of Hb S; (C) Concentration-dependent inhibition of SS cell sickling under hypoxia.
Figure 5
Figure 5
Binding of a pair of 5HMF molecules (cyan sticks) at the α-cleft of R2-state Hb. The α-chain (α1- and α2-subunits) colored in grey and β-chain (β1- and β2-subunits) colored in yellow. Water molecules are red spheres.
Figure 6
Figure 6
Binding of a pair of 5HMF-NO molecules (green sticks) at the α-cleft of T-state Hb. The α-chain (α1- and α2-subunits) colored in grey and β-chain (β1- and β2-subunits) colored in yellow. Water molecules are red spheres.
See this image and copyright information in PMC

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