Author ORCID Identifier
https://orcid.org/0000-0002-9898-4129
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Dr.Martin Safo
Abstract
Background: Sickle Cell Disease (SCD) is a genetic blood disorder impacting 100,000 individuals in the US and millions globally. The estimated lifetime medical expenses associated with SCD are approximately $1.7 million. SCD is caused by a genetic mutation of βGlu6 in hemoglobin (Hb) to βVal6, forming sickle Hb (Hb S). Hb S molecules polymerize when they release oxygen, resulting in sickling of red blood cells (RBCs) that stick to the walls of blood vessels, causing blockage, vaso-occlusive crisis, pain, hemolytic anemia and organs damage. Aromatic aldehydes have emerged as promising therapeutic agents for SCD. The compounds offer notable advantages, including their potential to modify Hb and increase the oxygen affinity of Hb S, resulting in inhibition of Hb S polymerization and RBCs sickling. Voxelotor is the first aromatic aldehyde approved by the FDA in 2019 for the treatment of SCD. Its pharmacologic effect of inhibiting RBCs sickling is solely due to increasing the O2 affinity of Hb (O2-dependent antisickling mechanism). This mechanism poses a potential on-target adverse effect by preventing the release of O2 from Hb to tissues if the therapeutic dose is increased to the level required to prevent vaso-occlusive crises. Voxelotor was recently withdrawn from the market due to low efficacy/risk ratio, underscoring the need for alternative therapeutic agents. Nonetheless, Voxelotor has provided proof of concept that aromatic aldehydes could serve as viable treatment options for SCD, paving the way for further research into similar therapeutic agents. The objective of this research was to design and synthesize benzamide-substituted aromatic aldehydes that not only prevent RBCs sickling by increasing the O2 affinity of Hb S but directly destabilize the polymer, leading to enhanced antisickling activity. These compounds were expected to be less susceptible to hydrolysis than their ester analogs, thus enhancing partitioning into RBCs and reducing potential toxicity.
Methods: Benzamide-substituted analogs of the previously reported lead compounds PP-10 and PP-14 were designed and synthesized and investigated for their in vitro ability to modify normal human Hb. Ten compounds (LA-2, LA-3, LA-4, LA-5, LA-6, LA-7, LA-8, LA-9, LA-11, and LA-12) synthesized in this study, along with two others synthesized by a lab mate (LA-1 and LA-10). The compounds were studied in vitro to evaluate their abilities to inhibit RBCs sickling, modify Hb into the high-oxygen affinity form, and evaluate their cytotoxic effects. PP-14 and/or VZHE-039, two previously studied polymer destabilizers were used as positive controls. The crystal structures of the compounds, including LA-1, LA-2, LA-3, LA-4, LA-5, LA-9 and LA-12 in complex with liganded Hb were determined. LA-1, which demonstrated very high antisickling activity, was selected for preliminary in vivo studies using Sprague-Dawley male rats via oral gavage at a dose of 100 mg/kg (n=6). Finally, kinetic binding studies were performed with LA-1, LA-7 and LA-12 to determine their binding affinity to Hb.
Results and Discussion: Ten LA compounds were synthesized with yields ranging from 20% to 60% and were fully characterized using 1H-NMR, 13C-NMR, mass spectrometry, and infrared spectroscopy. Their purity, determined by HPLC, was found to be greater than 95%. The ten compounds, in addition to two others synthesized by a lab mate (LA-1 and LA-10) when studied for their in vitro pharmacodynamic effects showed to modify Hb (33-100%) compared to the positive control VZHE-039 (98%) or PP-14 (100%), inhibit RBCs sickling under hypoxic conditions (26-88%) compared to the positive VZHE-039 (82%) or PP-14 (99%) at 2 mM. As expected from their design, several of the compounds demonstrated antisickling activity under anoxic conditions (7-89%) compared to (79%) by VZHE-039 at 2 mM, indicative of direct polymer destabilization. Similar to VZHE-039 and PP-14, the compounds increased the O2 affinity of Hb, which explains their antisickling activity under hypoxic conditions. The compounds showed no significant in vitro cytotoxic effect and partitioned highly into RBCs. Unexpectedly, during the in vivo study of LA-1 (oral administration to rats), the amide moiety of the compound (as previously observed with the ester moiety of PP-14) metabolized into its carboxylic acid metabolite, which may potentially pose problem with off-target adverse effect. Like PP-14 and VZHE-039, the crystal structures showed the LA compounds bound at the α -cleft to make Schiff-base interaction with αVal1 amine, as well as several hydrogen-bond and hydrophobic interactions with the protein that stabilize the R-state Hb, explaining the compounds ability to increase Hb affinity for O2 and prevent Hb S polymerization and RBCs sickling. Additionally, some of the LA compounds made water-mediated interaction with the surface-located αF-helix residues of Hb, explaining the compounds anoxic antisickling activities. Selected compounds (LA-1, LA-7 and LA-12), when tested for their equilibrium dissociation constants with Hb under steady-state and kinetic conditions show varying binding affinities for Hb. LA-1 and LA-12 with the highest affinity for Hb (KD of 0.11 mM) expectedly exhibited the highest Hb modification and/or sickling inhibition. The differences in the compounds’ functional and/or biological activities appear to correlate with the size of the amide substitution on the benzene ring. In general, compounds with smaller bulky side chain show the most potent activities and those with the largest side chain exhibit the lowest activities, the latter due to steric hindrance.
Conclusions: The designed compounds differ from their previously studied ester analogs by substituting the ester moiety with amide moiety. The compounds showed no significant cytotoxic effect, partitioned very well into RBCs, modify Hb to increase its O2 affinity with concomitant hypoxia sickling inhibition. Some of the compounds also showed significant polymer destabilization effect by preventing RBCs sickling under anoxia condition, due to interaction with the αF-helix as demonstrated by the crystal structures. In conclusion, even though these novel compounds showed very promising in vitro pharmacologic effect, their propensity to hydrolyze into the carboxylic analogs in vivo may pose potential adverse effect. Overall, the study serves as a model for future drug development and therapeutic advancements for SCD.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-10-2024