Development of nonretinoid rpe65 inhibitors for the potential treatment of dry age-related macular degeneration and stargardt disease

Abstract

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the industrialized world among individuals aged 65 years or older. The dry (atrophic) form of AMD, which accounts for approximately 85–90% of all cases, is characterized by the progressive accumulation of cytotoxic bisretinoid lipofuscin fluorophores, most notably A2E (N-retinylidene-N-retinylethanolamine), within the retinal pigment epithelium (RPE). A2E accumulation is directly linked to the flux of retinaldehyde intermediates through the canonical retinoid visual cycle, and its formation is exacerbated in Stargardt disease, the most common inherited macular dystrophy, which is caused by loss-of-function mutations in the ABCA4 gene. A critical and rate-limiting step in the visual cycle is the isomerohydrolase (IMH) reaction catalyzed by the retinal pigment epithelium-specific 65 kDa protein (RPE65), which converts all-trans-retinyl palmitate to 11-cis-retinol. Pharmacological modulation of RPE65 activity thus represents a rational strategy to reduce bisretinoid formation and slow disease progression. Prior work in the Cioffi laboratory, initially conducted at the Albany College of Pharmacy and Health Sciences (ACPHS) and subsequently continued at Rensselaer Polytechnic Institute (RPI), identified CU239 as a selective, competitive, and nonretinoid inhibitor of RPE65 (IC50 = 6 μM; KD = 231 nM). Through iterative medicinal chemistry optimization, a lead compound designated RPE65-71 was identified that demonstrated a 3-fold improvement in potency (IC50 = 2.1 μM), reduced molecular weight, decreased lipophilicity, and lower topological polar surface area relative to CU239, while lacking the metabolic liabilities associated with the clinically investigated RPE65 inhibitor (R)-emixustat. The present thesis describes the continuation of this drug discovery program at RPI. Building upon the RPE65-71 scaffold, four novel nonretinoid RPE65 inhibitor analogues were designed and synthesized: RPI-RPE65-56, RPI-RPE65-57, RPI-RPE65-58, and RPI-RPE65-59. Three of the four target analogues (RPI-RPE65-57, RPI-RPE65-58, RPI-RPE65-59) represent a new structural class derived from the arylacetamide pharmacophore, in which a 3-hydroxyphenylacetic acid scaffold is elaborated with diverse ether appendages at the meta position. RPI-RPE65-56 retains the thiazole core and introduces a 4-hydroxyphenethylamino modification. All compounds were synthesized via concise, modular routes and fully characterized by proton nuclear magnetic resonance (1H NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and high-performance liquid chromatography (HPLC). Biological evaluation of the four newly synthesized analogues was conducted in collaboration with Wake Forest University, yielding IC50 values of 32,568 ± 1 nM (RPI-RPE65-56), 56,029 ± 2 nM (RPI-RPE65-57), 141,656 ± 5 nM (RPI-RPE65-58), and 23,441 ± 2 nM (RPI-RPE65-59). Although all four analogues exhibited reduced potency relative to RPE65-71, the data provide valuable structure-activity relationship information that will guide the next iterative medicinal chemistry cycle. Melanin binding studies are ongoing in collaboration with Columbia University.