Biology subjects

Shashikadze, B.

Publications and source records attributed to Shashikadze, B..

4 recordsLinked to original sources

bioRxiv2024

Unbiased mapping of cereblon neosubstrate landscape by high-throughput proteomics

Molecular glue degraders (MGDs) are small molecules that harness the ubiquitin-proteasome system to induce degradation of target proteins, including those lacking conventional druggable pockets. Given the challenges in their rational design, MGD discovery predominantly relies on screening-based approaches, such as cell viability assays. However, one potential limitation of such screening methods is the risk of overlooking non-essential neosubstrates of potential therapeutic value. To address this concern, we present a high-throughput proteome-wide MGD screening platform utilizing label-free, data-independent acquisition mass spectrometry (DIA-MS) for integrated proteomics and ubiquitinomics analysis. Processing a diverse set of 100 CRBN-ligands across two cancer cell lines reveals a broad array of neosubstrates, including 50 novel candidates validated by MS-based ubiquitinomics. These findings considerably expand the current landscape of CRBN-mediated neosubstrates. Comprehensive hit validation and structure-degradation relationship analyses guided by global proteomics, identifies highly selective and potent phenyl glutarimide-based degraders of novel neosubstrates, including KDM4B, G3BP2 and VCL, none of which contain the classical CRBN degron motif. This study demonstrates that comprehensive, high-throughput proteomic screening offers new opportunities in MGD drug discovery.

biochemistry↗

bioRxiv2023

Gene Therapy for Cardiomyopathy associated with Duchenne Muscular Dystrophy in a Pig Model

BackgroundGenetic cardiomyopathies caused by mutations in the dystrophin gene (DMD) are only partially responsive to current pharmacological heart failure treatments, although dilated and arrhythomogenic phenotypes of cardiomyopathy are frequent. ObjectiveIn this study, we tested whether a normalization of Ca2+-handling by forced expression of SERCA2a in cardiomyocytes mitigates heart failure and arrhythmogenesis in a pig model for Duchenne muscular dystrophy (DMD). Methods and resultsMale offspring of pigs lacking DMD exon 52 are characterized by heart failure with reduced ejection fraction (HFrEF, EF 34.5{+/-}1.8% vs. 49.2{+/-}1.0% in control hearts), arrhythmogenesis due to large apical regions of reduced voltage amplitude and sudden cardiac death with a lifespan of usually less than 4 months. Slow antegrade intracoronary infusion of AAV1.SERCA2a (3x1013 virus genomes (vg) per pig) improved left ventricular ejection fraction (EF 47.3{+/-}2.0%, p<0.05) to a similar extent as germline editing of DMD{Delta}52 to DMD{Delta}51-52, inducing a Becker dystrophy (BMD) genotype (EF 46.7{+/-}3.8%). Moreover, AAV.SERCA2a significantly reduced myocardial inflammation and fibrosis and areas of reduced AP amplitude. ConclusionsIn DMD pigs, 3x1013vg/heart of GMP-grade AAV1.SERCA2a sufficed to normalize left ventricular function and improved electrical vulnerability of the heart. Hence, AAV.SERCA2a may serve as a treatment option for DMD cardiomyopathy.

physiology↗

bioRxiv2023

Increased pulmonary monocyte infiltration and attenuated phagocytosis defines perinatal dysfunction of innate immunity in Cystic Fibrosis

In Cystic Fibrosis (CF) patients, cycles of infection and inflammation lead to fatal lung damage. While diminished mucus clearance is restored by highly effective CFTR modulator therapy, inflammation and infection persist in treated patients, suggesting alternative mechanisms may contribute to impaired immunity in CF. Here, we made use of a CF pig model to investigate the innate immune system at birth, before the onset of infection. We observed a substantial change in the composition of tissue resident immune cells towards emergency myelopoiesis, accompanied by increased infiltration of monocytes into CF lungs. A more immature status in the transcriptome profile of classical monocytes in CF pigs and preschool children with CF correlated with reduced phagocytic capacity, confirming a congenital and translationally conserved aberration of the immune system in CF. The lack of CFTR expression in circulating monocytes indicates an indirect etiology of these effects and suggests that additional immunological treatments are necessary for CF patients. One Sentence SummaryIncreased infiltration of lung tissue by monomyeloid cells and their impaired phagocytic potential cause dysfunctional imprinting of mucosal immunity in Cystic Fibrosis airways at birth and suggest early and specific treatment of the innate immune system in patients.

immunology↗

bioRxiv2021

A scalable, clinically severe pig model for Duchenne muscular dystrophy

Large animal models for Duchenne muscular dystrophy (DMD) are crucial for preclinical evaluation of novel diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMD{Delta}52) resemble molecular, clinical and pathological hallmarks of DMD, but cannot be propagated by breeding due to death before sexual maturity. Therefore, female DMD+/- carriers were generated. A single founder animal had 11 litters with 29 DMDY/-, 34 DMD+/- as well as 36 male and 29 female wild-type (WT) offspring. Breeding with F1 and F2 DMD+/- carriers resulted in additional 114 DMDY/- piglets. The majority of them survived for 3-4 months, providing large cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance of human disease mechanisms. Importantly, DMDY/- pigs reveal progressive fibrosis of myocardium and increased expression of connexin-43, associated with significantly reduced left ventricular fractional shortening and ejection fraction already at age 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability of DMDY/- pigs. Our breeding cohort of DMD{Delta}52 pigs and standardized tissue repositories from DMDY/- pigs, DMD+/- carriers, and WT littermate controls provide important resources for studying DMD disease mechanisms and for testing novel diagnostic procedures and treatment strategies.

pathology↗