MODEL-BASED IN-VITRO PK/PD PROFILING OF NOVEL SYNTHETIC ALLOSTERIC EFFECTORS OF HEMOGLOBIN (AEH) AS POTENTIAL SICKLE CELL DISEASE (SCD) THERAPEUTICS

Introduction: Allosteric effectors of hemoglobin (AEH) represent a class of synthetic aromatic aldehydes that transiently form covalent interactions (Schiff-base) with hemoglobin (Hb) to form Hb-AEH adduct, preventing the HbS polymerization and sickling of red blood cells (RBC). The overall objective of this research was to aid in the optimization of novel AEH by understanding their target-site disposition of AEH in relevant biological matrices, e.g., HbA solution, whole blood (WB) and human liver cytosol (HLC), a surrogate of aldehyde dehydrogenase (ALDH)-mediated oxidative metabolism. Methods: A “universal” HPLC-UV/Vis assay method was developed for the quantitation of HbA-AEH adduct for chemically different AEH in HbA solution and in WB. Steady-state (SS) concentration-dependence and time- dependence HbA binding were characterized by a sigmoidal Bmax-model (KD) and a kinetic binding model (kon, koff). The Warburg method was used to assess the enzymatic oxidation of selected AEH in HLC. WB disposition of 5-HMF (VCU lead, terminated in Phase 2) and VZHE-039 (current top candidate, pre-IND) was assessed by measuring their HbA-AEH adduct in WB and plasma exposures. Results: All vanillin-derived benzaldehydes exhibit enhanced binding affinity in HbA solution, primarily due to their faster kon. Across AEH, i.e., benzaldehydes and furaldehydes studied, kon and koff values are strongly (log-log) correlated (r=0.993, n=7), suggesting that the molecular modifications of the current AEH scaffold enhance their interactions with Hb (as intended), but also (inadvertently) increase their Hb-AEH adduct dissociation. In HLC, some AEH showed oxidative metabolism (CLint: 5-HMF \u3c TD-7 \u3c INN-310 \u3c acetaldehyde, prototypical substrate). Presence of the meta-methoxy group (in TD-7) favors ALDH-mediated oxidation, while the ortho-hydroxy group (in VZHE-039) protects the aldehyde group/AEH from metabolism. In WB, Hb-AEH adduct concentrations for VZHE-039 are sustained in WB for 24 hrs, confirming its resistance to ALDH metabolism in RBC. A less than proportional increase in plasma cmax, but proportional AUC along with prolonged tmax with the initial concentration, [VZHE-039]0, were observed for VZHE-039, indicating rate-limiting, but ultimately complete dissolution, prior to any HbA binding. The estimated apparent binding affinity (1/KDkinetic’) for VZHE-039 in WB is 23-fold lower than observed in HbA solution, primarily due to a 300-fold slower apparent kon’. On the other hand, HbA-AEH adduct concentrations for 5-HMF in WB decrease after 1.5 hrs - thus providing conclusive evidence of rapid intra-RBC ALDH-mediated metabolism - which resulted in an 8-fold lower apparent binding affinity (KD’ of 3.0 mM) from concentration-dependency study at 1.5 hrs in WB. However, after accounting for first-order metabolism in the WB disposition model, the KDkinetic’ for 5-HMF in WB was quite similar to that in HbA solution; dissociation of 5-HMF from Hb was slow and determined the terminal decline of HbA-AEH adduct in WB. Conclusions: WB disposition of AEH is determined by the kinetics of three concurrent processes, namely (saturable) Hb binding, (non-saturable) ALDH-mediated metabolism in RBC, as well as reversible, first-order PPB/RBC membrane binding. Although the KD value for VZHE-039 is 3-fold lower than 5-HMF in HbA solution, both compounds exhibit similar apparent KD’ values in WB, albeit due to different mechanisms, namely ALDH-mediated metabolism for 5-HMF and extensive PPB/RBC membrane binding for VZHE-039 – both processes reducing AEH concentrations at the target site, Hb. Concurrent PPB/RBC membrane binding for lipophilic benzaldehydes, e.g., VZHE-039, also slows down their overall WB HbA adduct formation, but helps sustain them. Key molecular features of AEH to enhance HbA binding and reduce intra-RBC metabolism have been identified, allowing further rational design of more potent (in WB) drug candidates for further druggability evaluation - with VZHE-039 as the top candidate

Chern-Simons, Wess-Zumino and other cocycles from Kashiwara-Vergne and associators

Descent equations play an important role in the theory of characteristic classes and find applications in theoretical physics, e.g. in the Chern-Simons field theory and in the theory of anomalies. The second Chern class (the first Pontrjagin class) is defined as $p= \langle F, F\rangle$ where $F$ is the curvature 2-form and $\langle \cdot, \cdot\rangle$ is an invariant scalar product on the corresponding Lie algebra $\mathfrak{g}$. The descent for $p$ gives rise to an element $\omega=\omega_3 + \omega_2 + \omega_1 + \omega_0$ of mixed degree. The 3-form part $\omega_3$ is the Chern-Simons form. The 2-form part $\omega_2$ is known as the Wess-Zumino action in physics. The 1-form component $\omega_1$ is related to the canonical central extension of the loop group $LG$. In this paper, we give a new interpretation of the low degree components $\omega_1$ and $\omega_0$. Our main tool is the universal differential calculus on free Lie algebras due to Kontsevich. We establish a correspondence between solutions of the first Kashiwara-Vergne equation in Lie theory and universal solutions of the descent equation for the second Chern class $p$. In more detail, we define a 1-cocycle $C$ which maps automorphisms of the free Lie algebra to one forms. A solution of the Kashiwara-Vergne equation $F$ is mapped to $\omega_1=C(F)$. Furthermore, the component $\omega_0$ is related to the associator corresponding to $F$. It is surprising that while $F$ and $\Phi$ satisfy the highly non-linear twist and pentagon equations, the elements $\omega_1$ and $\omega_0$ solve the linear descent equation