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For humans to see, the retinoid (visual) cycle converts incident light entering in the eye into electrochemical signals that can be transmitted to the brain as neuronal impulses. The chemical reactions that take place within the retinoid cycle allows the regeneration of key intermediates to allow for vision to take place. Dysregulation of these chemical processes along with gene mutations is associated with disease. These diseases often lead to an alteration in vision causing blindness. Age-related macular degeneration is an example of the dysregulation within the retinoid cycle. The 3-dimensional binding site of bovine RPE65 to an exogenous substrate (''R'')-emixustat shown in '''Figure 1A''' and '''Figure 1B'''. '''Figure 1C''' shows the surface of bovine RPE65 where (''R'')-emixustat would enter to access the internal binding pocket of RPE65. Both '''Figure 1B''' and '''Figure 1C''' was generated using PyMol. (''R'')-emixustat is a drug candidate that was tested for the treatment of dry age-related macular degeneration.  

[[Image:RPE65_Emixustat_and_Palmitate_PyMOL.jpg|thumb|center|512 px|alt=Figure 1B: Emixustat and palmitate ester in Binding Pocket| '''Figure 1B:''' Bovine RPE65 Binding Pocket with Emixustat and palmitate ester generated in PyMOL using PDB: ''4RSV'']]

[[Image:Binding_surface_overlap.jpg|thumb|center|512 px|alt=Figure 1C: Emixustat RPE65 binding pocket opening| '''Figure 1C:''' Bovine RPE65 Opening into Binding Pocket with the electron mesh of Emixustat outlined using PyMOL, PDB: ''4RSV'']]

Following dissociation from opsin, all-''trans''-retinal is transported from the lumen of the photoreceptor disk though an ATP-cassette transporter 4 (''ABCA4''). The enzyme all-''trans''-retinol dehydrogenase (atRDH) reduces all-''trans''-retinal into all-''trans''-retinol. Another transporter enzyme, interphotoreceptor retinoid-binding protein (IRBP) facilitates the transport of all-''trans''-retinol back into the hRPE. Within the hRPE, all-''trans''-retinol is esterified by the enzyme lecithin retinol acyltransferase (LRAT) to form all-''trans''-retinyl ester. RPE65 also known as retinoid isomerohydrolase (IMH) then converts all-''trans''-retinyl ester into 11-''cis''-retinol and palmityl acid. Following the RPE65 catalyzed reaction, 11-''cis''-retinol dehydrogenase (11-''cis''-RDH) oxidizes 11-''cis''-retinol into 11-''cis''-retinal. 11-''cis''-retinal is finally transported back into the photoreceptors to be conjugated to opsin to form the rhodopsin complex. This process is shown in Figure 2. <ref>DOI 10.1016/j.bbadis.2018.04.014</ref>

[[Image:Visual_Cycle_RPE65.jpg|thumb|center|512 px|alt=Figure 2: Human Visual Cycle| '''Figure 2:''' A schematic representation of the Retinoid (Visual) Cycle in Humans. (Abbreviations: all-''trans''-ROL, all-''trans''-retinol; LRAT, lecithin retinol acyltransferase; RPE65, retinal pigment epithelium 65; 11-''cis''-ROL, 11-''cis''-retinol; 11-''cis''-RDH, 11-''cis''-retinol dehydrogenase; 11-''ci''s-RAL, 11-''cis''-retinal; ''hV'', light energy; all-''trans''-RAL, all-''trans''-retinal; atRDH, all-''trans''-retinol dehydrogenase) <ref> DOI 10.1016/j.bbadis.2018.04.014 </ref>]]

Crystal structure of bovine RPE65 (PDB: ''3FSN''), which is 99% similar to human RPE65, (although the crystal structure for human RPE65 is not currently available) is used as the basis of studying RPE65 structure. RPE65 resembles a seven-bladed β-propeller with single-stranded extension on blades VI and VII and two-stranded extension on blade III shown in '''Figure 4'''. The top face of the β-propeller is defined by connecting the outer strand of the β-sheet with the inner strand of the next β-sheet. The iron cofactor is located near the top surface of the propeller which is coordinated by four His residues and three secondary Glu residues. Each blade of the propeller contributes one His residue to coordinate with the iron cofactor. A hydrophobic tunnel leads the protein exterior to the active site which is defined by the iron ion to accommodate the passage of retinoids (which are conjugated to a fatty acid tail) from the membrane to the RPE65 catalytic site. The mouth of the tunnel is surrounded by three groups of nonpolar residues that contribute to the overall hydrophobicity of the tunnel and the integration with the lipid bilayer. There are also a few aromatic amino acid side chains that reside in this portion of the enzyme. This suggest that the depth of RPE65 membrane is restricted to the proximal portions of the phospholipid acyl chains with respect to the polar head groups. Arg and Lys residues within this region also contribute to the association with the negatively charged phospholipid head groups. <ref> DOI 19805034 </ref>

[[Image:RPE65_Figure4_S7_blades.jpg|thumb|center|512 px|alt=Figure 4: RPE65 structure| '''Figure 4:''' Crystal structure of bovine RPE65 viewed from the bottom face of the seven-bladed β-propeller labeled in roman numerals from I to VII <ref> DOI 19805034 </ref>]]

[[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val. The bond length interactions are shown in angstroms from the iron atom. <ref> DOI 19805034 </ref>]]

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> Using Cluster Omega, the amino acid sequence of NinaB and bovine RPE65 was compared. Although the structure alignment is quite different, there are still some sequence homology between the two enzymes that may suggest an ancestor linkage.  

Comparing bovine RPE65 across different species using a BLAST search showed that RPE65 is highly conserved between animals. From mammals, to rodents there was at least a 97% similarity in sequences. As an example shown below in '''Figure 7A''' is a sequence alignment between bovine RPE65 and hamster RPE65. Throughout the sequence alignment there are only 12 amino acid residues that are different between the two species. By showing RPE65 is conserved, researchers are able to use bovine RPE65 to predict the structure and activity of human RPE65 in drug testing and drug development. '''Figure 7B''' shows the lineage of all know RPE65 enzymes.

[[Image:sequence_alignment.jpg|thumb|center|512 px|alt=Figure 7A: bovine and hamster RPE65 sequence alignment| '''Figure 7A:''' Sequence Alignment between Bovine RPE65 and hamster RPE65 showing highly conserved amino acid residue between organisms.]]

[[Image:Lineage_RPE65_Full.jpg|thumb|center|512 px|alt=Figure 7B: Full RPE65 lineage| '''Figure 7B:''' RPE65 lineage tree using BLAST. Note that the red arrow is a continuation of the lineage tree after "multiple organisms" and is not to scale. Note that the scale changes from 0.01 to 0.006 between the two portions of the lineage]]

Mutations within the RPE65 enzyme can impact the enzyme catalyzed reactions that is pertinent to the retinoid cycle. Disease such as Leber congenital amaurosis (LCA) or retinal dystrophy disease such as retinitis pigmentosa (RP) arise due to the mutations in RPE65. Often these diseases can lead to impaired vision or even blindness. Since human and bovine RPE65 share approximately 99% of the amino acid sequences, it can be assumed that the secondary and tertiary structures are similar. This relationship allows us to map human LCA and RP to the bovine RPE65 amino acid sequences. In LCA and RP patients, it was noted that most of the amino acid mutations were adjacent to the blades of the β-propeller rather than in the connecting loops and helices. Blade VII was the most common area of mutation. Since mutations here would alter how the β-propeller seals into its proper structure, this could impact the location and residues that contribute to chelating the iron ion. For patients with LCA, missense mutations caused a major change in the first and second shell of the iron ion ligands which is essential for RPE65 isomerase activity. As such patients with these mutations had non-functional RPE65 enzymes. Amino acid Arg91, Tyr368 and His182 were most often mutated in patients with RPE-65 associated LCA or RP. This showed that these amino acid positions were integral to the function of the enzymatic activity. Mutations such as His182Arg or Tyr368His caused major structural deformations that altered the accommodation of the substituted side chain. In such cases, enzyme activity was decreased or lost. Arg91 is known to be important in positioning RPE65 to the membrane-binding elements. Arg91 also forms a salt bridge with Glu127 which is located on the C-terminal side of the enzyme. This interaction is critical for subcellular localization. As a result, mutations here alteres the ability of RPE65 for subcellular localization. <ref> DOI 19805034 </ref>

RPE65 interacts with other ligands to catalyze an enzymatic isomerhydrolase activity.  

The endogenous ligand for human RPE65 is all-''trans''-retinyl ester. Binding to the active site as well as the catalytic mechanism were mentioned above in the sections ''[1.2.2.2] Active Site Structural Analysis of RPE65'' and ''[1.2.3] Proposed Enzymatic Mechanism of RPE65'' respectively.  

Emixustat (ACU-4429) shown in '''Figure 8''' 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 8:''' (''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 9''').

[[Media:RPE65_with_emixustat.mp4|Figure 9: 3-dimensional structure of (''R'')-emixustat bound in RPE65 with bond lengths ]]

Dry AMD represents the progressive neurodegenerative disorder that impact the specialize neurons within the central part of the retinal called the macula. The degeneration of choriocapillaires, hRPE and the neurosensory cells such as rods and cones photoreceptor can lead to sensory loss within the macula. Age-dependent accumulation of cytotoxic lipofuscin such as A2E within the hRPE can lead to irreversible damage in the retina. It is suggested that retinoid cycle modulation can help prevent the accumulation of lipofuscin in the hRPE. Since RPE65 is expressed exclusively within the hRPE cells, this makes RPE65 a potential for drug research and development as theoretically a drug targeting RPE65 would minimize off-target effects. RPE65 is responsible for the chemical conversion of all-trans-retinyl ester to 11-cis-retinol which is the rate limiting step, in the retinoid cycle. As such, modulating RPE65 would effectively modulate the entire retinoid cycle. <ref> DOI 19668560 </ref>

Although (''R'')-emixustat failed to show clinical outcomes due to significant pharmacokinetic and pharmacodynamic limitations, (''R'')-emixustat became a proof-of-concept for RPE65 inhibition as well as the basis for structure-based drug design for future RPE65 inhibitors. Using (''R'')-emixustat, researchers were able to develop a screening for other RPE65 inhibitors. As part of the drug discovery process, subsequent hit identification, hit to lead, lead optimization processes would be required to develop new line of RPE65 inhibitors. These processes would require the use of structure activity relationships as well as structural property relationships to overcome the shortfalls that emixustat presented.