DOI
https://doi.org/10.25772/2HWS-NF55
Defense Date
2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Martin Safo
Second Advisor
Yan Zhang
Abstract
Sickle cell disease(SCD) is the most common inherited hematologic disorder, affecting about 100,000 people in the U.S., and over 15 million worldwide. The disease occurs as a result of a single-point mutation of glutamic acid (bGlu6) in normal hemoglobin (HbA) to valine (bVal6) forming sickle hemoglobin (HbS). This mutation leads to polymerization of HbS and sickling of red blood cells (RBCs) when exposed to low oxygen (O2) tensions. Polymerization of deoxygenated sickle Hb (deoxyHbS), initiated by an interaction between β2Val6 from one deoxyHbS molecule and a hydrophobic acceptor pocket in an adjacent deoxyHbS molecule is the principal cause of the pathophysiology associated with the disease. This primary pathophysiology leads to a cascade of secondary adverse effects, e.g. adhesion of sickled RBCs to tissue endothelium, oxidative stress, inflammation, painful vaso-occlusive crisis, and eventually chronic endothelial and organ damage. The primary interaction of the HbS polymer is further stabilized by several secondary interactions, e.g. those involving aAsn78 from a surface-located aF-helix. Clinical management of SCD is centered around pain relief, prophylaxis against the infection and periodic transfusion to reduce the concentration of sickle RBCs in the bloodstream. Hydroxyurea, an approved drug that induces HbF production, was the only drug widely used and remains the current “gold standard” for SCD treatment. However, poor compliance in some patients due to side effects, tends to limit its use. Three other drugs have also been recently approved, including L-glutamine, Crizilazumab and GBT-440 (Voxelotor). GBT-440 was the first of a new class of aromatic aldehyde-containing compounds that interfere with HbS polymerization by increasing Hb- O2 affinity.
Compounds with aromatic aldehydes are known to bind HbS by forming Schiff-base interaction with the N-terminal α-subunit Val1 amines of HbS, increasing hemoglobin (Hb) affinity for O2 and prevent the hypoxia-induced HbS polymerization. These compounds have been studied for their potential to treat SCD. With the recently approved compound GBT-440, other compounds with aromatic aldehydes, e.g. VZHE-039 and PP14 discovered by our group are also undergoing preclinical studies for the treatment of SCD. These compounds, in addition to their ability to increase Hb affinity for O2 , prevent HbS polymerization by directly interrupting the secondary interactions at the surface-located aF-helix of the protein with a concomitant antisickling effect, an action that is independent of the presence of O2. This additional antisickling effect of (VZHE-039 and PP14) has been found to provide a synergistic effect along with the increase in Hb-O2 affinity which might translate into a powerful molecule to treat SCD. As these lead compounds (VZHE-039 and PP14) shed the light on an exciting antisickling mechanism (O2-independent antisickling effect), a major objective of our research is to develop aromatic aldehydes that not only exhibit dual antisickling effects (O2-dependent and O2-independent antisickling effects) like VZHE-039 and PP14, but also are expected to exhibit the same level of potency of the FDA approved aromatic aldehyde (GBT-440). Based on the chemical structures of GBT-440, VZHE-039, and PP14, we designed compounds in silico that we hypothesize will bind Hb with a single molecule like GBT-440, a property responsible for GBT-440’s high potency, and also interact and perturb a surface-located αF-helix like VZHE-039 or PP14, a property responsible for the O2-independent antisickling effect. Seven novel aromatic aldehydes (noted as RA compounds) were successfully synthesized. One of the compounds (RA7), has structural similarity to GBT-440 with an isopropyl substituted pyrazole on the pyridine ring, while the other six compounds (RA1, RA2, RA3, RA4, RA5 and RA6) have an unsubstituted 5-membered ring on the pyridine ring. All compounds were tested for their antisickling potencies, using sickle whole blood (SS) under 2.5% O2 , which is a measure of O2-dependent antisickling effect, and 100% N2, which is a measure of O2-independent antisickling effect. Hb modification and P50 shift were also performed using aliquot samples from the antisickling studies to quantify the Hb-compound adduct formation (Schiff-base formation) and to test the ability of the compounds to increase Hb-O2 affinity using HPLC analysis and a HemoxTM Analyzer, respectively. A time-dependent Hb modification study to monitor the metabolic stability of the compounds over the course of 6 hours was also performed. To establish the mode of interaction with Hb, we further conducted x-ray crystallographic studies with selected compounds to understand the biological activities and mode of binding of the compounds.
The compounds showed a concentration-dependent O2-dependent antisickling effect that directly correlated with the increase in Hb-O2 affinity and modification of Hb, but unexpectedly not as potent as the positive controls GBT-440 or VZHE-039, with the exception RA7 that showed comparable antisickling potency with GBT-440. Unexpectedly, none of the compounds showed an antisickling effect under 100% nitrogen compared to VZHE-039, suggesting a lack of O2-independent antisickling activity and consequently, lack of dual antisickling property. Consistently, and similar to GBT-440, the crystallographic study showed the RA compounds did not make close interactions with the aF-helix as anticipated, presumably explaining the compound’s lack of O2-independent antisickling effect. Interestingly, while RA7, the only compound with structural similarity and comparable potency to GBT-440, binds Hb in a 1:1 ratio, the other compounds which lack isopropyl group on the 5-membered ring, bind Hb in a 2:1 ratio. The significantly low potency of RA1, RA2, RA3, RA4, RA5 and RA6 is due to poor binding to Hb as indicated by their structural studies. Similar to VZHE-039 and GBT-440, the compounds showed metabolic stability over the course of a 6-hours experiment.
The second objective of our study was to improve the pharmacokinetic (PK) property of our previously studied potent methoxy derivative antisickling compound, TD-7 which showed poor oral bioavailability due to rapid in-vivo oxidative metabolism of the aldehyde group. In this second project, we derivatized TD-7 into a thiazolidine ethyl ester prodrug (Pro-7), that we expected would protect the aldehyde from quick metabolism and overcome its metabolic instability. Pro-7 showed good stability in in-vitro studies. Furthermore, unlike TD-7, Pro-7 showed a slow onset but increasing Hb modification, P50 shift and antisickling activities that were sustained over the course of the time-dependent experiment. In-vivo results showed that Pro-7 is capable of hydrolyzing to release TD-7, however, the blood concentration of TD-7 did not reach an ideal therapeutic level.
Although the objective to design and discover small molecules that not only exhibit dual antisickling effects, but also exhibit the same level of potency as GBT-440 did not translate into the expected outcomes, we expect to use the totality of the results to aid in targeted structural modification for improved biological properties of these compounds. Our study also provided a proof of concept that the thiazolidine prodrug strategy can be applied to aromatic aldehydes to improve their metabolic stability which may lead to an improved drug candidate.
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Is Part Of
VCU Theses and Dissertations
Date of Submission
8-6-2022