Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

The impact of nanobodies on GPCR structural biology and their potential as therapeutic agents.

The family of human G protein-coupled receptors (GPCRs) is comprised of about 800 different members, with about 35% of current pharmaceutical drugs targeting GPCRs. However, GPCR structural biology, necessary for structure-guided drug design, has lagged behind that of other membrane proteins, and it was not until the year 2000 when the first crystal structure of a GPCR (rhodopsin) was solved. Starting in 2007, the determination of additional GPCR structures was facilitated by protein engineering, new crystallization techniques, complexation with antibody fragments, and other strategies. More recently, the use of camelid heavy-chain-only antibody fragments (nanobodies) as crystallographic chaperones has revolutionized the field of GPCR structural biology, aiding in the determination of more than 340 GPCR structures to date. In most cases, the GPCR structures solved as complexes with nanobodies (Nbs) have revealed the binding mode of cognate or non-natural ligands; in a few cases, the same Nb has acted as an orthosteric or an allosteric modulator of GPCR signaling. In this review we summarize the multiple ingenious strategies that have been conceived and implemented in the last decade to capitalize on the discovery of nanobodies to study GPCRs from a structural perspective. Significance Statement G protein-coupled receptors (GPCRs) are major pharmacological targets, and the determination of their structures at high resolution has been essential for structure-guided drug design and for insights about their functions. Single domain antibodies (nanobodies) have greatly facilitated the structural determination of GPCRs, by forming complexes directly with the receptors or indirectly through protein partners.

Structural basis for the allosteric modulation of rhodopsin by nanobody binding to its extracellular domain.

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.