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YC-001 rescues P23H opsin from the ER to the plasma membrane. a–c Chemical structures of 11-cis-retinal, 9-cis-retinal, and YC-001, respectively. The three chemical moieties of YC-001 are shaded and numbered. d. Diagram of the β-Gal fragment complementation assay used for the HTS. Briefly, two complementary fragments of β-Gal (EA and PK) were individually fused with a plasma membrane-anchored peptide, the pleckstrin homology domain of phospholipase C δ (PLC-EA, in cyan), and the mouse P23H-opsin mutant (P23H-PK, in magenta), respectively. A U2OS stable cell line was generated that co-expressed both PLC-EA and P23H-PK. Owing to its inherent instability, P23H-PK accumulated in the ER, whereas PLC-EA remained on the plasma membrane, leading to a loss of β-Gal activity due to the separation of the two fragments of this enzyme. Upon treatment with an active compound that rescues the folding and transport of P23H opsin to the plasma membrane, a recovery of β-Gal activity is observed due to co-localization of PK and EA. e The activities of YC-001 (black boxes) and 9-cis-retinal (magenta circles) were tested in a dose-dependent manner employing the β-Gal fragment complementation assay. Each compound was preincubated for 24 h before β-Gal activity was tested. Activity scores were standardized to the effect of 5 µM 9-cis-retinal as 100%. Dose dependence was fitted by the Hill function with Origin software. R², EC50 (μM), and Max score for each compound were obtained from curve fitting and are listed in the graph. The experiment was repeated three times. f Activities of 40 µM YC-001 (black boxes) and 5 µM 9-cis-retinal (magenta circles) were tested as a function of time with the β-Gal fragment complementation assay. The time course graph was fitted with a Hill function and T1/2s were obtained and listed in the graph. This experiment was repeated twice. g Activities of YC-001 together with 5 µM 9-cis-retinal were tested in a dose-dependent manner and plotted in black triangles. This experiment was repeated twice. The activity scores were plotted as the averages of three biological replicates, with the error bars as the s.d.s
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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...
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Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber’s congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical...
Citations
... Rod opsin mutations such as P23H often exhibit inherent protein instability leading to photoreceptor cell degeneration and autosomal dominant retinitis pigmentosa (adRP) 43,44 . The binding of ligands to GPCRs can stabilize protein folding via a pharmacological chaperone effect, often restoring proteostasis of mutant GPCRs, including Rho, δopioid, V2 vasopressin, and Frizzled 4 receptors [44][45][46][47] . ...
Rhodopsin is a prototypical G protein-coupled receptor (GPCR) critical for vertebrate vision. Research on GPCR signaling states has been facilitated using llama-derived nanobodies (Nbs), some of which bind to the intracellular surface to allosterically modulate the receptor. Extracellularly binding allosteric nanobodies have also been investigated, but the structural basis for their activity has not been resolved to date. Here, we report a library of Nbs that bind to the extracellular surface of rhodopsin and allosterically modulate the thermodynamics of its activation process. Crystal structures of Nb2 in complex with native rhodopsin reveal a mechanism of allosteric modulation involving extracellular loop 2 and native glycans. Nb2 binding suppresses Schiff base deprotonation and hydrolysis and prevents intracellular outward movement of helices five and six – a universal activation event for GPCRs. Nb2 also mitigates protein misfolding in a disease-associated mutant rhodopsin. Our data show the power of nanobodies to modulate the photoactivation of rhodopsin and potentially serve as therapeutic agents for disease-associated rhodopsin misfolding.
... As proof of concept, the delivery of VCP siRNA using reverse magnetofection in organotypic cultures of Rho P23H transgenic retinas effectively prevented photoreceptor cell death and attenuated retinal degeneration in vitro (Sen et al., 2021b). Other small molecules newly identified that rescued the transport of rhodopsin include the chaperon YC-001 which protected Abca4 −/− * Rdh8 −/− doublemutant mice from bright light-induced photoreceptor death (Chen et al., 2018). ...
Therapy development for neurodegenerative diseases of the retina constitutes a major unmet medical need, and this may be particularly relevant for inherited diseases of the retina, which are largely untreatable to this day. Therapy development necessitates appropriate models to improve the understanding of the underlying degenerative mechanisms, as well as for the testing and evaluation of novel treatment approaches. This review provides an overview of various in vitro model systems used to study retinal neuroprotection. The in vitro methods and technologies discussed range from primary retinal cell cultures and cell lines, to retinal organoids and organotypic retinal explants, to the cultivation of whole eyeballs. The advantages and disadvantages of these methods are compared and evaluated, also in view of the 3R principles (i.e., the refinement, reduction, and replacement of live animal testing), to identify suitable in vitro alternatives for in vivo experimentation. The article further expands on the use of in vitro models to test and evaluate neuroprotective treatments and to aid the development of retinal drug delivery systems. Among the pharmacological agents tested and characterized in vitro are such that interfere with aberrant cyclic guanosine monophosphate (cGMP) -signaling or such that inhibit the activities of poly (ADP-ribose) polymerase (PARP), histone deacetylases (HDAC), calpain-type proteases, as well as unfolded protein response-related stress. We then introduce nanoparticle-based drug delivery systems and discuss how different in vitro systems may be used to assess their efficacy in the treatment of retinal diseases. The summary provides a brief comparison of available in vitro models and relates their advantages and limitations to the various experimental requirements, for instance, for studies into disease mechanisms, novel treatments, or retinal toxicity. In many cases, combinations of different in vitro models may be required to obtain a comprehensive view of the efficacy of a given retinal neuroprotection approach.
... When investigating the slower pathways in a rhodopsin photocycle, iso-pigments would also be a proper alternative, except for mutants or analog pigments, where the photocascades may deviate [113,133,154]. When using iso-pigments in vitro or in cell culture, one must take into account the much slower incorporation rate of 9-cis retinal into the opsin, the lower thermodynamic stability, and the lower photosensitivity of iso-pigments [258][259][260][261][262]. In particular, when using 9-cis retinal in physiological studies-for instance, when scanning for chaperones stabilizing rhodopsin mutants, investigating rhodopsin oligomerization in vivo, or studying organoid differentiation-, it is essential to verify essential data with 11-cis retinal [263][264][265][266][267][268][269]. ...
Rhodopsin, the first visual pigment identified in the animal retina, was shown to be a photosensitive membrane protein containing covalently bound retinal in the 11-cis configuration, as a chromophore. Upon photoexcitation the chromophore isomerizes in femtoseconds to all-trans, which drives the protein into the active state. Soon thereafter, another geometric isomer—9-cis retinal—was also shown to stably incorporate into the binding pocket, generating a slightly blue-shifted photosensitive protein. This pigment, coined isorhodopsin, was less photosensitive, but could also reach the active state. However, 9-cis retinal was not detected as a chromophore in any of the many animal visual pigments studied, and isorhodopsin was passed over as an exotic and little-relevant rhodopsin analog. Consequently, few in-depth studies of its photochemistry and activation mechanism have been performed. In this review, we aim to illustrate that it is unfortunate that isorhodopsin has received little attention in the visual research and literature. Elementary differences in photoexcitation of rhodopsin and isorhodopsin have already been reported. Further in-depth studies of the photochemical properties and pathways of isorhodopsin would be quite enlightening for the initial steps in vision, as well as being beneficial for biotechnological applications of retinal proteins.
... We discovered a group of pharmacological chaperones of RHO, including nonretinoids YC-001 and F5257-0462 (26), by a cell-based high-throughput screening (HTS; refs. 27,28). ...
... Both YC-001 and F5257-0462 improve the glycosylation profile of RHO P23H and rescue multiple misfolded RHO mutants from ER to the plasma membrane, demonstrating the potent pharmacological chaperone activity of these compounds. Furthermore, we found YC-001 protects the retinae from light damage in Abca4 -/-, Rdh8 -/mice (26), an acute retinal degeneration model. However, the efficacy of these pharmacological chaperones in retina expressing misfolded RHO was not known. ...
... Our previous study suggests that YC-001 binds in the chromophore pocket of rod opsin (26). While the complex structure of rod opsin with YC-001 is challenging to obtain, we used a docking calculation by AutodockVina (see Supplemental Methods, ref. 33) to see how small-molecule chaperones may bind to rod opsin. ...
Rhodopsin (RHO)-associated retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified non-retinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules towards the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Further, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL) indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that non-retinoid chaperones are promising drug candidates in treating RHO-associated RP.
... (7) The expression of many of retinopathy variants can be partially restored by analogs of rhodopsin's native 11-cis-retinal cofactor and/ or other small molecules that bind and stabilize the opsin apoprotein. (12)(13)(14)(15)(16) Despite the discovery of numerous therapeutic lead compounds and the initiation of several clinical trials, there are currently no approved treatments for these disorders. An improved understanding of the molecular effects of the spectrum of clinical rhodopsin variants may help to identify a subset of "correctable" rhodopsin variants that could be targeted in future clinical trials. ...
... (33) As is true for other classes of correctors,(34) binding affinity is a key consideration for emerging retinoid and non-retinoid rhodopsin correctors. (10,14,35) Nevertheless, because the observed shifts are rooted in stability, the moderate class II variants that exhibit large shifts in response to 9-cis-retinal should generally represent the most favorable target variants for other correctors as well. ...
... Most efforts to discover correctors have been evaluated in relation to their effects on the expression and/ or maturation of the P23H variant-the most common pathogenic rhodopsin variant. (12)(13)(14)(15)36) However, P23H is also among the most poorly expressed variants and exhibits only modest sensitivity to retinal (Table S1). Based on this consideration, ongoing corrector screens could potentially achieve better sensitivity by targeting moderate class II variants that exhibit a greater response (i.e. ...
Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9- cis -retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. 67 of these apparent class II variants exhibit a measurable increase in expression in the presence of 9- cis -retinal. Nevertheless, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts in relation to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (ΔN73, S131P, and R135G) and show that two retain residual function in vitro . Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.
... T17M, P23H, and E181K [18]. Other two studies reported on the discovery of small chaperones able to bind bovine rod opsin and to promote membrane localization of P23H opsin though with EC 50 comparable or worse than that of 9-cis-retinal [35,36]. Specifically: i) the discovered small chaperones are non-retinoids; ii) the compound YC-001, discovered by cell-based high-throughput screening, showed inverse agonist and non-competitive antagonist activities towards rod opsin [35]; and iii) the compound S-RS1 inferred from virtual and thermofluor screening was found to stabilize rod opsin by binding to the orthosteric side [36]. ...
... Other two studies reported on the discovery of small chaperones able to bind bovine rod opsin and to promote membrane localization of P23H opsin though with EC 50 comparable or worse than that of 9-cis-retinal [35,36]. Specifically: i) the discovered small chaperones are non-retinoids; ii) the compound YC-001, discovered by cell-based high-throughput screening, showed inverse agonist and non-competitive antagonist activities towards rod opsin [35]; and iii) the compound S-RS1 inferred from virtual and thermofluor screening was found to stabilize rod opsin by binding to the orthosteric side [36]. ...
... Future applications of the computational protocol include the structure-based optimization of the 13-cis-5,8-ERA chaperone and the discovery/design of non-retinoid small chaperones with translational potential as therapeutic agents for the cure of adRP linked to rod opsin mutations. The discovery/design of nonretinoid chaperones will take into account the information on two completely unrelated chemical scaffolds published almost simultaneously to our chaperone [35,36]. Furthermore, the approach is exportable to other conformational diseases linked to protein missense mutations. ...
Failure of a protein to achieve its functional structural state and normal cellular location contributes to the etiology and pathology of heritable human conformational diseases. The autosomal dominant form of retinitis pigmentosa (adRP) is an incurable blindness largely linked to mutations of the membrane protein rod opsin. While the mechanisms underlying the noxious effects of the mutated protein are not completely understood, a common feature is the functional protein conformational loss. Here, the wild type and 39 adRP rod opsin mutants were subjected to mechanical unfolding simulations coupled to the graph theory-based protein structure network analysis.
A robust computational model was inferred and in vitro validated in its ability to predict endoplasmic reticulum retention of adRP mutants, a feature linked to the mutation-caused misfolding. The structure-based approach could also infer the structural determinants of small chaperone action on misfolded protein mutants with therapeutic implications.
The approach is exportable to conformational diseases linked to missense mutations in any membrane protein.
... On the other hand, there are a relevant number of works in which no-photoswitchable small molecules have been used in cellular and animal models of RP. These classes of molecules are related or not with retinal, and principally act as molecular chaperones able to restore a correct Rho folding and transport/localization, especially in the pathogenic variants P23H and T17M, rescuing light sensitivity [139][140][141][142][143]. The discussion of these molecules is out of the scope of this review. ...
In the last decade, innovative therapeutic strategies against inherited retinal degenerations (IRDs) have emerged. In particular, chemical- and opto-genetics approaches or a combination of them have been identified for modulating neuronal/optical activity in order to restore vision in blinding diseases. The ‘chemical-genetics approach’ (optopharmacology) uses small molecules (exogenous photoswitches) for restoring light sensitivity by activating ion channels. The ‘opto-genetics approach’ employs light-activated photosensitive proteins (exogenous opsins), introduced by viral vectors in injured tissues, to restore light response. These approaches offer control of neuronal activities with spatial precision and limited invasiveness, although with some drawbacks. Currently, a combined therapeutic strategy (optogenetic pharmacology) is emerging. This review describes the state of the art and provides an overview of the future perspectives in vision restoration.
... Results are expressed as mean ± standard error (s.e.m) for animal studies while the other results are expressed as mean ± standard deviation (s.d.) 62 . The sample size for the animal studies was validated by Gpower3 software using the post-hoc power analysis for a twotailed t-test 68,69 . The effect size index was calculated by the equation: ...
Developing injectable antibacterial and conductive shape memory hemostatic with high blood absorption and fast recovery for irregularly shaped and noncompressible hemorrhage remains a challenge. Here we report injectable antibacterial conductive cryogels based on carbon nanotube (CNT) and glycidyl methacrylate functionalized quaternized chitosan for lethal noncompressible hemorrhage hemostasis and wound healing. These cryogels present robust mechanical strength, rapid blood-triggered shape recovery and absorption speed, and high blood uptake capacity. Moreover, cryogels show better blood-clotting ability, higher blood cell and platelet adhesion and activation than gelatin sponge and gauze. Cryogel with 4 mg/mL CNT (QCSG/CNT4) shows better hemostatic capability than gauze and gelatin hemostatic sponge in mouse-liver injury model and mouse-tail amputation model, and better wound healing performance than Tegaderm™ film. Importantly, QCSG/CNT4 presents excellent hemostatic performance in rabbit liver defect lethal noncompressible hemorrhage model and even better hemostatic ability than Combat Gauze in standardized circular liver bleeding model.
The retina is part of the central nervous system specialized for vision. Inherited retinal diseases (IRD) are a group of clinically and genetically heterogenous disorders that lead to progressive vision impairment or blindness. Although each disorder is rare, IRD accumulatively cause blindness in up to 5.5 million individuals worldwide. Currently, the pathophysiological mechanisms of IRD are not fully understood and there are limited treatment options available. Most IRD are caused by degeneration of light-sensitive photoreceptors. Genetic mutations that abrogate the structure and/or function of photoreceptors lead to visual impairment followed by blindness caused by loss of photoreceptors. In healthy retina, photoreceptors structurally and functionally interact with retinal pigment epithelium (RPE) and Müller glia (MG) to maintain retinal homeostasis. Multiple IRD with photoreceptor degeneration as a major phenotype are caused by mutations of RPE- and/or MG-associated genes. Recent studies also reveal compromised MG and RPE caused by mutations in ubiquitously expressed ciliary genes. Therefore, photoreceptor degeneration could be a direct consequence of gene mutations and/or could be secondary to the dysfunction of their interaction partners in the retina. This review summarizes the mechanisms of photoreceptor-RPE/MG interaction in supporting retinal functions and discusses how the disruption of these processes could lead to photoreceptor degeneration, with an aim to provide a unique perspective of IRD pathogenesis and treatment paradigm. We will first describe the biology of retina and IRD and then discuss the interaction between photoreceptors and MG/RPE as well as their implications in disease pathogenesis. Finally, we will summarize the recent advances in IRD therapeutics targeting MG and/or RPE.
Simple Summary
Surgery, radiotherapy, and chemotherapy, the standard oncological treatments, are increasingly revealing their limitations. Population treatments are based on the percentage of patients that benefit, with no guarantee of sustained treatment effectiveness even when personalized and molecularly targeted therapies are considered. Cancer always develops with the “consent“ of the immune system, which is considered the guardian of the cancer process. Therapy aimed at tumor destruction does not recognize the subtleties associated with the likely coexistence of tumor tissue protection systems. A new concept of regulatory therapy, based on targeted interference in the mechanisms of molecular processes underlying carcinogenesis along with cancer promotion and progression, may represent a promising direction in the search for effective therapies. Recently, there has been increased interest in exosomes, which are the cellular products of intercellular communication and carriers of signaling molecules, based on the potential for regulatory interference in cancer process. We have knowledge about these mechanisms, but do we have the technology to allow such interference? If so, the time has come for regulatory therapy in oncology.
Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles, together with apoptotic bodies form a diverse group of nanoparticles that play a crucial role in intercellular communication, participate in numerous physiological and pathological processes. In the context of cancer, they can allow the transfer of bioactive molecules and genetic material between cancer cells and the surrounding stromal cells, thus promoting such processes as angiogenesis, metastasis, and immune evasion. In this article, we review recent advances in understanding how EVs, especially exosomes, influence tumor progression and modulation of the microenvironment. The key mechanisms include exosomes inducing the epithelial–mesenchymal transition, polarizing macrophages toward protumoral phenotypes, and suppressing antitumor immunity. The therapeutic potential of engineered exosomes is highlighted, including their loading with drugs, RNA therapeutics, or tumor antigens to alter the tumor microenvironment. Current techniques for their isolation, characterization, and engineering are discussed. Ongoing challenges include improving exosome loading efficiency, optimizing biodistribution, and enhancing selective cell targeting. Overall, exosomes present promising opportunities to understand tumorigenesis and develop more targeted diagnostic and therapeutic strategies by exploiting the natural intercellular communication networks in tumors. In the context of oncology, regulatory therapy provides the possibility of reproducing the original conditions that are unfavorable for the existence of the cancer process and may thus be a feasible alternative to population treatments. We also review current access to the technology enabling regulatory intervention in the cancer process using exosomes.
