Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Bruton’s Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.

No abstract

Tebipenem pivoxil is the first orally available carbapenem antibiotic and has been approved in Japan for treating ear, nose, and throat and respiratory infections in pediatric patients. Its active moiety, tebipenem, has shown potent antimicrobial activity in vitro against clinical isolates of Enterobacterales species from patients with urinary tract infections (UTIs), including those producing extended-spectrum β-lactamases (ESBLs) and/or AmpC β-lactamase.

Disrupting protein‐protein interactions is difficult due to the large and flat interaction surfaces of the binding partners. The BLIP and BLIP‐II proteins are unrelated in sequence and structure and yet each potently inhibit β‐lactamases. High‐throughput oligonucleotide synthesis was used to construct a 12,470‐member library containing overlapping linear and cyclic peptides ranging in size from 6 to 21 amino acids that scan through the sequences of BLIP and BLIP‐II. Phage display affinity selections and deep sequencing revealed that, despite the differences in interaction surfaces with β‐lactamases, rapid enrichment of consensus peptide regions originating from both BLIP and BLIP‐II contact residues in the binding interface occurred. BLIP and BLIP‐II peptides that were enriched by affinity selection were shown to bind β‐lactamases and disrupt the BLIP/β‐lactamase interaction. The results suggest that peptides that bind at and disrupt PPI interfaces can be identified through systematic peptide library construction, affinity selection, and deep sequencing.

Human noroviruses (HuNoV) are the leading cause of acute gastroenteritis worldwide. The humoral immune response plays an important role in clearing HuNoV infections and elucidating the antigenic landscape of HuNoV during an infection can shed light on antibody targets to inform vaccine design. Here, we utilized Jun-Fos-assisted phage display of a HuNoV genogroup GI.1 genomic library and deep sequencing to simultaneously map the epitopes of serum antibodies of six individuals infected with GI.1 HuNoV. We found both unique and common epitopes that were widely distributed among both nonstructural proteins and the major capsid protein. Recurring epitope profiles suggest immunodominant antibody footprints among these individuals. Analysis of sera collected longitudinally from three individuals showed the presence of existing epitopes in the pre-infection sera, suggesting these individuals had prior HuNoV infections. Nevertheless, newly recognized epitopes surfaced seven days post-infection. These new epitope signals persisted by 180 days post-infection along with the pre-infection epitopes, suggesting a persistent production of antibodies recognizing epitopes from previous and new infections. Lastly, analysis of a GII.4 genotype genomic phage display library with sera of three persons infected with GII.4 virus revealed epitopes that overlapped with those identified in GI.1 affinity selections, suggesting the presence of GI.1/GII.4 cross-reactive antibodies. The results demonstrate that genomic phage display coupled with deep sequencing can characterize HuNoV antigenic landscapes from complex polyclonal human sera to reveal the timing and breadth of the human humoral immune response to infection.