DOI
https://doi.org/10.25772/H7MZ-6D76
Defense Date
2024
Document Type
Thesis
Degree Name
Master of Science
Department
Pharmaceutical Sciences
First Advisor
Martin Safo
Abstract
ABSTRACT
UTILIZING HEMOGLOBIN BINDING KINETICS AND X-RAY CRYSTALLOGRAPHY IN SICKLE CELL DISEASE DRUG DEVELOPMENT
By Ahmed Alghamdi, M.Sc. in Medicinal Chemistry
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Medicinal Chemistry at Virginia Commonwealth University.
Virginia Commonwealth University, 2024.
Director: Dr. Martin K. Safo Professor, Department of Medicinal Chemistry
Introduction: Sickle Cell Disease (SCD) is characterized by severe hemolytic anemia, recurrent painful crises, and chronic organ damage, posing significant challenges to patients' quality of life and longevity. While current FDA-approved therapies offer symptomatic relief, they do not address the underlying molecular mechanisms driving the disease. Aromatic aldehydes have emerged as promising anti-sickling agents, with compounds like the recently approved SCD drug Voxelotor demonstrating efficacy in enhancing hemoglobin (Hb) oxygen affinity and inhibiting HbS polymerization and red blood cell sickling. This study uses a universal HPLC-UV/Vis assay and X-ray crystallography to aid in understanding the biological activities of antisickling aromatic aldehydes, and to inform lead selections and/or further structural modification for improving the pharmacologic activities of the compounds. The ultimate goal of this research is to develop novel therapeutic agents for SCD that can effectively prevent HbS polymerization and its associated pathophysiology.
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Methods A "universal" HPLC-UV/Vis assay method was employed to quantify the HbA-AEH adduct for the chemically diverse aromatic aldehydes or AEHs (CAT-059, CAT- 180, CAT-181, CAT-191, and/or CAT-120) in a concentration-dependent and time-dependent manners. A sigmoidal Bmax model was fit to the steady-state HbA-AEH adduct concentration – AEH0 profiles by nonlinear regression (Scientist®) characterized with maximal HbA-AEH adduct formed (Bmax), equilibrium dissociation constant (K ss), and Hill coefficient (n). A bimolecular interaction (AEH:Hb = 1:1) model was fit to the HbA-AEH adduct concentration- time profiles across [AEH]0 using Scientist® to estimate the forward rate constant (kon) and backward rate constant (koff), as well as dissociation constant (KDkinetics) and equilibrium half- life (t1/2eq). X-ray crystallography was employed to determine the atomic structures of liganded
Hb in complex with the aromatic aldehydes CAT-059, CAT-180, CAT-181, CAT-194A and CAT-187).
Result and Discussion: The study investigated the HbA binding kinetics and atomic interactions of various aromatic aldehydes with Hb, focusing on compounds CAT-059, CAT- 180, CAT-181, CAT-191, CAT-120, CAT-194, CAT-194A, and/or CAT-187. These
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compounds have previously been studied for their functional and biological activities, including their RBC sickling inhibitory properties. The tested compounds demonstrated diverse HbA binding kinetics, with K SS and K Kinetic values reflecting equilibrium dissociation constants in steady-state and kinetic conditions, respectively. The compounds are also bound to the α-cleft of Hb, explaining their mechanisms of actions on atomic level.
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CAT-120 exhibited the highest binding affinity for Hb (K kinetic ≈ 0.068 mM) among the tested compounds, including the positive control VZHE-039 (K kinetic = 0.10 mM, which is attributed to the addition of a second aldehyde group that likely allows the compound to cross-link the α-cleft to make Schiff-base interactions with both α-subunits Val1 amines. In contrast, VZHE-039 has only one aldehyde moiety and makes Schiff-base interaction with one α-subunits Val1 amine. Consistently, CAT-120 exhibits the most potent antisickling activity among the compounds. Unfortunately, we could not obtain the crystal structure of CAT-120 to experimentally determine its mode of binding. It thus appear that compounds with two aldehyde moiety that could potentially cross-link the two alpha-subunits may offer superior pharmacologic activity.
Interestingly, removal of the methylhydroxyl substituent on the pyridine ring of
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VZHE-039, as in CAT-059, did not lead to loss of affinity (K kinetic of 0.10 mM and 0.08 mM, respectively), emphasizing that such substituents may not be necessary for biological potency.
This was made obvious by the crystal structure of CAT-059 that showed that even though it lacks methylhydroxyl group, the pyridine nitrogen atom is involved in several strong water- mediated interactions, mostly with the αF-helix of the protein. Consistently, VZHE-039 and CAT-059 showed similar biological activities. The interactions with the αF-helix also explains this compound’s ability to inhibit RBC sickling independent of oxygen, that is through polymer destabilization.
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Substitution of the methylhydroxyl moiety on the pyridine ring of VZHE-039 with amide moiety to form CAT-180 and CAT-181 only moderately affected the compounds affinity (K kinetic of 0.11 and 0.13 mM, respectively vs 0.10 mM for VZHE-039). However, the amide CAT-191 showed significantly weaker affinity for Hb (K kinetic = 0.26 mM). The crystal structures of CAT-180 and CAT-181 with amide substitutions on the pyridine ring make similar interactions with Hb as VZHE-039, explaining their similar affinity and biological potency, including their ability to interact with the F-helix and destabilize the polymer for O2-independent sickling inhibition. Although the crystal structure of CAT-191 was not determined, the observed low Hb affinity is likely due to steric hindrance because of the bulky nature (butyl amide) of the amide moiety. Consistently, CAT-187 with even more bulkier side chain of CAT-191 showed significantly weaker electron density signifying weak affinity for Hb.
CAT-194A, an acid analog of the amide CAT-194 binds similarly as VZHE-039, explaining this compound biological similarity with VZHE-039. The crystallographic study revealed that, unlike Voxelotor, all compounds bind two molecules to the Hb tetramer with liganded Hb, stabilizing the relaxed state Hb and increasing oxygen affinity.
Conclusion: In conclusion, these diverse compounds not only enhance Hb affinity for oxygen but also directly destabilize the polymer by interacting with the F-helix, offering an O2- independent antisickling activity. By focusing on novel synthetic AEH candidates and employing both binding kinetics and X-ray crystallography studies, this research elucidated critical insights into their binding parameters and atomic interactions with Hb. Structural modifications, such as the addition of a second aldehyde group, were found to enhance binding affinity, while the introduction of substituents on the pyridine ring did not significantly impact pharmacologic activity. Larger or bulkier substituted moiety on the pyridine ring reduced affinity. The correlation between binding kinetics and structural analysis underscores the importance of molecular design in developing effective AEH compounds. Overall, this study represents a promising approach toward developing more effective treatments for SCD, providing a blueprint for future drug development efforts and therapeutic innovations.
Rights
© Ahmed K. Alghamdi
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
3-26-2024