Selective Protein of Interest Degradation through the Split-and-Mix Liposome Proteolysis Targeting Chimera Approach.

Proteolysis Targeting Chimera (PROTAC) technology represents a promising new approach for target protein degradation using a cellular ubiquitin-proteasome system. Recently, we developed a split-and-mix nanoplatform based on peptide self-assembly, which could serve as a self-adjustable platform for multifunctional applications. However, the lower drug efficacy limits further biomedical applications of peptide-based SM-PROTAC. In this study, we develop a novel split-and-mix PROTAC system based on liposome self-assembly (LipoSM-PROTAC), concurrent with modification of FA (folate) to enhance its tumor-targeting capabilities. Estrogen receptors (ERα) were chosen as the protein of interest (POI) to validate the efficacy of Lipo degraders. Results demonstrate that this PROTAC can be efficiently and selectively taken up into the cells by FA receptor-positive cells (FR+) and degrade the POI with significantly reduced concentration. Compared to the peptide-based SM-PROTACs, our designed LipoSM-PROTAC system could achieve therapeutic efficacy with a lower concentration and provide opportunities for clinical translational potential. Overall, the LipoSM-based platform shows a higher drug efficacy, which offers promising potential applications for PROTAC and other biomolecule regulations.

Versatile Split-and-Mix Liposome PROTAC Platform for Efficient Degradation of Target Protein In Vivo.

PROTAC (Proteolysis TArgeting Chimeras) is a promising therapeutic approach for targeted protein degradation that recruits an E3 ubiquitin ligase to a specific protein of interest (POI), leading to its degradation by the proteasome. Recently, we developed a novel split-and-mix PROTAC system based on liposome self-assembly (LipoSM-PROTAC) which could achieve target protein degradation at comparable concentrations comparable to small molecules. In this study, we expanded protein targets based on the LipoSM-PROTAC platform and further examined its therapeutic effects in vivo. Notably, this platform could efficiently degrade the protein level of MEK1/2 in A375 cells or Alk in NCI-H2228 cells and display obvious tumor inhibition (60-70% inhibition rate) with negligible toxicity. This study further proved the LipoSM-PROTAC's application potentials.

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

Identification of an unknown frameshift variant of NOG in a Han Chinese family with proximal symphalangism.

Proximal symphalangism (SYM1) is an autosomal dominant disorder manifested by ankylosis of the proximal interphalangeal joints of fingers, carpal and tarsal bone fusion, and conductive hearing loss in some cases. Herein, we clinically diagnosed a Chinese patient with fusions of the bilateral proximal interphalangeal joints in the 2-5 digits without conductive hearing loss. Family history investigation revealed that his mother and grandfather also suffered from SYM1. Whole exome sequencing was performed to detect the genetic lesion of the family. The candidate gene variants were validated by Sanger sequencing. By data filtering, co-segregation analysis and bioinformatics analysis, we highly suspected that an unknown heterozygous frameshift variant (c.635_636insG, p.Q213Pfs*57) in NOG was responsible for the SYM1 in the family. This variant was predicted to be deleterious and resulted in a prolonged protein. This finding broadened the spectrum of NOG mutations associated with SYM1 and contributed to genetic diagnosis and counseling of families with SYM1.

The Novel Compound Heterozygous Mutations of ECEL1 Identified in a Family with Distal Arthrogryposis Type 5D.

INTRODUCTION: Distal arthrogryposis type 5D (DA5D) is an autosomal recessive disease. The clinical symptoms include contractures of the joints of limbs, especially camptodactyly of the hands and/or feet, unilateral ptosis, a round-shaped face, arched eyebrows, and micrognathia, without ophthalmoplegia. ECEL1 is a DA5D causative gene that encodes a membrane-bound metalloprotease. ECEL1 plays important roles in the final axonal arborization of motor nerves in limb skeletal muscles and neuromuscular junction formation during prenatal development. METHODS: A DA5D family with webbing of the elbows and fingers was recruited. We performed whole-exome sequencing (WES) and filtered mutations by disease-causing genes of arthrogryposis multiplex congenita (AMC). Mutational analysis and cosegregation confirmation were then performed. RESULTS: We identified novel compound heterozygous mutations of ECEL1 (NM_004826: c.69C>A, p.C23∗ and c.1810G>A, p.G604R) in the proband. CONCLUSIONS: We detected causative mutations in a DA5D family, expanding the spectrum of known ECEL1 mutations and contributing to the clinical diagnosis of DA5D.

Compound heterozygous GNPTAB mutations cause mucolipidosis II or III alpha/beta in two Chinese families.

OBJECTIVE: Mucolipidosis II and III alpha/beta (ML II & ML III alpha/beta) are rare autosomal recessive lysosomal storage disorders. ML II is clinically evident from birth with a progressive course and fatal outcome in childhood. The typical phenotypes of ML II include limited statural growth, craniofacial abnormality, skeletal malformation, intelligence developmental deficiency and visceral organ abnormality. ML III is milder than ML II. Mutations in GNPTAB cause the ML II/III. METHODS: Two families with ML II/III (initially undiagnosed) were recruited. We applied whole-exome sequencing (WES) and filtered mutations by genes causing lysosomal storage diseases with skeletal involvement. Mutational analysis and co-segregation confirmation were then performed. RESULTS: We presented two families with ML II or ML III alpha/beta. By WES, the compound heterozygosity of GNPTAB (c.2404C>T, p.Q802* and c.2590dup, p.E864Gfs*4) is identified in a family with ML II, and c.1364C>T, p.A455V and c.2715+1G>A are detected in a family with ML III alpha/beta. CONCLUSION: We detected the causative mutations in two ML II/III families by WES and confirmed their diagnosis of the diseases. The present identification of mutations expands the spectrum of known GNPTAB mutations and it may contribute to novel approaches to genetic diagnosis and counseling for patients with ML II/III.