Quantitative assessment of the in-vitro binding kinetics of antisickling aromatic aldehydes with hemoglobin A: A universal HPLC-UV/Vis method to quantitate Schiff-base adduct formation

  • J Pharm Biomed Anal. 2023 Jan 20:223:115152. doi: 10.1016/j.jpba.2022.115152.
Xiaomeng Xu  1 ,  Mohini S Ghatge  2 ,  Boshi Huang  2 ,  Ahmed Alghamdi  2 ,  Huiqun Wang  2 ,  B Daniel Pierce  3 ,  Osheiza Abdulmalik  4 ,  Yan Zhang  2 ,  Martin K Safo  5 ,  Jürgen Venitz  6
Affiliations
  • 1. Division of Cardiometabolic and Endocrine Pharmacology, Office of Clinical Pharmacology, Center of Drug Evaluation and Research, US FDA, Silver spring, MD 20993, USA; Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA.
  • 2. Department of Medicinal Chemistry and The Institute for Structural Biology, Drug Discovery and Development, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA.
  • 3. Department of Biology, University of Richmond, Richmond, VA 23173, USA.
  • 4. Division of Hematology, The Children's Hospital of Philadelphia, PA 19104, USA.
  • 5. Department of Medicinal Chemistry and The Institute for Structural Biology, Drug Discovery and Development, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA. Electronic address: [email protected].
  • 6. Department of Pharmaceutics, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA.
PMID: 36399908 DOI: 10.1016/j.jpba.2022.115152
Abstract

Aromatic aldehydes act as allosteric effectors of Hemoglobin (AEH), forming Schiff-base adducts with the protein to increase its oxygen (O2) affinity; a desirable property in sickle cell disease (SCD) treatment, as the high-O2 affinity Hemoglobin (Hb) does not polymerize and subsequently prevents erythrocytes sickling. This study reports the development, validation, and application of a weak cation-exchange HPLC assay - quantifying the appearance of Hb-AEH adduct - as a "universal" method, allowing for the prioritization of AEH candidates through an understanding of their Hb binding affinity and kinetics. Concentration- and time-dependent Hb binding profiles of ten AEHs were determined with HPLC, followed by the appropriate non-linear modeling to characterize their steady-state binding affinity (KDss), and binding kinetics second-order association (kon) and first-order dissociation (koff) rate constants. Vanillin-derived AEHs exhibited enhanced binding affinity to Hb, primarily due to their faster kon. Across AEH, kon and koff values are strongly correlated (r = 0.993, n = 7), suggesting that modifications of the AEH scaffold enhanced their interactions with Hb as intended, but inadvertently increased their Hb-AEH adduct dissociation. To our knowledge, the present study is the first to provide valuable insight into Hb binding kinetics of antisickling aromatic aldehydes, and the assay will be a useful platform in screening/prioritizing drug candidates for SCD treatment.

Keywords
Allosteric effectors of hemoglobin; Antisickling; Binding affinity and kinetics; Hemoglobin; Sickle cell disease; Universal method.
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