Sandbox Reserved 895: Difference between revisions

From an evolutionary standpoint it might be expected that the RPE65 catalytic mechanism would resemble that of the carotenoid oxygenase as sequence homology places RPE65 in the same family as the CCOs, this is not the case. Although the current enzymatic mechanism for RPE65 is only hypothesized with current research, there is not enough experimental evidence to definitively rule out that RPE65 catalyzes its function with molecular oxygen in the isomerization reaction. However, if such were the case the reaction would require even more complex chemistry which is not justifiable by the current scientific evidence. A study by Oberhauser and colleagues in 2008 on NinaB, a member of the CCO family from moths with isomerooxygenase activity was described, indicating that the members of this family originally developed isomerase activity while keeping the carotenoid oxygenase activity. The results of this study suggested a functional link between vertebrate RPE65 and insect NinaB which are both essential for the synthesis of key visual cycle chromophores. The function of NinaB showed that RPE65, a retinoid isomerase in the CCO family, retained the emergent activity that was found in a common ancestor. Comparing the topologies between the enzyme and substrate between NinaB and RPE65 showed that the iron cofactor was not directly involved in the double bond isomerization activity. Although further research would be needed to determine the ancestry of RPE65, it is fascinating to study the evolution and the diversification of protein family in animals by following the trace from a single multifunctional ancestral protein to several highly specialized enzymes seen in vertebrates. <ref> DOI 19020100 </ref>

Emixustat (ACU-4429) shown in '''Figure 7''' is an investigational small molecule inhibitor of RPE65 first invented by a British-American chemist, Ian L. Scott. When synthesized, emixustat usually presents of a racemic mixture of (''R'')-emixustat and (''S'')-emixustat. The (''R'')-isomer is associated with increased binding affinity and potency and is used in drug development and research. Formulated as a hydrochloride salt, (''R'')-emixustat hydrochloride is taken by mouth and functions as a visual cycle modulator (VCM) in atrophic (dry) age related macular degeneration (AMD). (''R'')-emixustat has been shown to reduce toxic retinal byproducts in the retinoid cycle such as N-retinylidiene-N-retinylethanolamine (A2E).  

[[Image:Figure7_Emixustat.jpg|thumb|center|512 px|alt=Figure 7: Emixustat| '''Figure 7:''' (''R'')-Emixustat]]

Zhang and colleagues determined the binding interactions between RPE65 and emixustat by looking at the protein-ligand complex under three different considerations. The three considerations were RPE65 with a racemic emixustat, RPE65 with pure (''R'')-emixustat and RPE65 with pure (''S'')-emixustat. With all three cases, residual maps showed the presence of bound palmitate in the active site with the carboxylate oxygen forming a monodentate coordinate bond with the iron ion. The hydroxyl moiety in emixustat interacted via a hydrogen bond with the hydroxyl moiety in Thr147 whereas the primary amine was involved in a polar interaction with the carboxylate groups of Glu148 as well as the bound palmitate ligand. Crystal structure that was obtained in the presence of the racemic mixture of emixustat showed that the electron density was consistent with exclusive binding of the (''R'')-isomer ('''Figure 1B'''). Using the pure (''R'')-isomer and the pure (''S'')-isomer as a follow up study to the experiment confirmed the stereoselectivity for the (''R'')-isomer of emixustat. Stereoselectivity was present because of the interaction between the hydroxyl moieties in the enantiomers. Looking at the electron density of the 3-amino-1-phenylpropan-1-ol moiety of (''R'')-emixustat showed that when the racemic mixture was used the binding was that of the (''R'')-isomer. This showed that (''R'')-emixustat had a higher binding affinity when compared to (''S'')-emixustat. The difference in binding affinity and potency was likely because of the less favorable polar interaction observed in the (''S'')-isomer. Bond lengths for the hydroxyl-Thr137 Oγ , amine-Glu148 Oε2 and amine-palmitate O1 interactions were 3.1, 3.2 and 2.8 angstroms for the (''S'')-isomer and 3, 2.7 and 2.6 angstroms for the (''R'')-isomer respectively ('''Figure 8''').

 

The difference in bond lengths also confirms the increase in binding affinity and potency of the (R)-isomer. These interactions can be seen in '''Figure 9''' . <ref> DOI 26075817 </ref>

[[Image:Figrue9_R_S_Emixustat_binding_pocket.jpg|thumb|center|512 px|alt=Figure 9ABCD: R and S Emixustat Binding Pocket| '''Figure 9:''' (A) Circular Dichroism on R, the (''R'')-isomer; M, the Racemic mixutre; and S, the (''S'')-isomer (B) Racemic emixustat bound to the RPE65 binding pocket (C) (''R'')-emixustat bound to the RPE65 binding pocket (D) (''S'')-emixustat bound to the RPE65 binding pocket <ref> DOI 26075817 </ref>]]