ABSTRACT
Rhodopsin homeostasis is tightly coupled to rod photoreceptor cell survival and vision. Mutations resulting in the misfolding of rhodopsin can lead to autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration that currently is untreatable. Using a cell-based high-throughput screen (HTS) to identify small molecules that can stabilize the P23H-opsin mutant, which causes most cases of adRP, we identified a novel pharmacological chaperone of rod photoreceptor opsin, YC-001. As a non-retinoid molecule, YC-001 demonstrates micromolar potency and efficacy greater than 9-cis-retinal with lower cytotoxicity. YC-001 binds to bovine rod opsin with an EC50 similar to 9-cis-retinal. The chaperone activity of YC-001 is evidenced by its ability to rescue the transport of multiple rod opsin mutants in mammalian cells. YC-001 is also an inverse agonist that non-competitively antagonizes rod opsin signaling. Significantly, a single dose of YC-001 protects Abca4 -/- Rdh8 -/- mice from bright light-induced retinal degeneration, suggesting its broad therapeutic potential.
Subject(s)
Neuroprotective Agents/pharmacology , Protein Folding/drug effects , Retinal Degeneration/drug therapy , Retinal Rod Photoreceptor Cells/drug effects , Rhodopsin/metabolism , Thiophenes/pharmacology , ATP-Binding Cassette Transporters/genetics , Alcohol Oxidoreductases/genetics , Animals , Cell Line, Tumor , Disease Models, Animal , Diterpenes , Female , HEK293 Cells , High-Throughput Screening Assays , Humans , Light/adverse effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , NIH 3T3 Cells , Neuroprotective Agents/therapeutic use , Protein Transport/drug effects , Protein Transport/genetics , Retinal Degeneration/etiology , Retinal Degeneration/pathology , Retinal Rod Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/pathology , Retinal Rod Photoreceptor Cells/radiation effects , Retinaldehyde/pharmacology , Retinaldehyde/therapeutic use , Rhodopsin/agonists , Rhodopsin/antagonists & inhibitors , Rhodopsin/genetics , Thiophenes/therapeutic use , Treatment OutcomeABSTRACT
Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.
Subject(s)
Hydrazones/pharmacology , Naphthalenes/pharmacology , Ribonucleotide Reductases/antagonists & inhibitors , Salicylates/pharmacology , Catalytic Domain , Cell Cycle/drug effects , Crystallography, X-Ray , Deoxyadenine Nucleotides/metabolism , Drug Screening Assays, Antitumor/methods , Humans , Hydrazones/chemistry , Naphthalenes/chemistry , Ribonucleoside Diphosphate Reductase , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/metabolism , Salicylates/chemistry , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolismABSTRACT
Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.
