An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations.

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 Mpro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

Ultrafast distant wound response is essential for whole-body regeneration.

Injury induces systemic responses, but their functions remain elusive. Mechanisms that can rapidly synchronize wound responses through long distances are also mostly unknown. Using planarian flatworms capable of whole-body regeneration, we report that injury induces extracellular signal-regulated kinase (Erk) activity waves to travel at a speed 10-100 times faster than those in other multicellular tissues. This ultrafast propagation requires longitudinal body-wall muscles, elongated cells forming dense parallel tracks running the length of the organism. The morphological properties of muscles allow them to act as superhighways for propagating and disseminating wound signals. Inhibiting Erk propagation prevents tissues distant to the wound from responding and blocks regeneration, which can be rescued by a second injury to distal tissues shortly after the first injury. Our findings provide a mechanism for long-range signal propagation in large, complex tissues to coordinate responses across cell types and highlight the function of feedback between spatially separated tissues during whole-body regeneration.

Ultrafast and long-range coordination of wound responses is essential for whole-body regeneration.

Injury induces systemic, global responses whose functions remain elusive. In addition, mechanisms that rapidly synchronize wound responses through long distances across the organismal scale are mostly unknown. Using planarians, which have extreme regenerative ability, we report that injury induces Erk activity to travel in a wave-like manner at an unexpected speed (∻1 mm/h), 10-100 times faster than those measured in other multicellular tissues. This ultrafast signal propagation requires longitudinal body-wall muscles, elongated cells forming dense parallel tracks running the length of the organism. Combining experiments and computational models, we show that the morphological properties of muscles allow them to minimize the number of slow intercellular signaling steps and act as bidirectional superhighways for propagating wound signals and instructing responses in other cell types. Inhibiting Erk propagation prevents cells distant to the wound from responding and blocks regeneration, which can be rescued by a second injury to distal tissues within a narrow time window after the first injury. These results suggest that rapid responses in uninjured tissues far from wounds are essential for regeneration. Our findings provide a mechanism for long-range signal propagation in large and complex tissues to coordinate cellular responses across diverse cell types, and highlights the function of feedback between spatially separated tissues during whole-body regeneration.

Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy.

Recent advances in DNA synthesis technology have made it possible to rewrite the entire genome of an organism. The major hurdles in this process are efficiently identifying and fixing the defect-inducing sequences (or "bugs") during rewriting. Here, we describe a high-throughput, semiquantitative phenotype assay for evaluating the fitness of synthetic yeast and identifying potential bugs. Growth curves were measured under a carefully chosen set of testing conditions. Statistical analysis revealed strains with subtle defects relative to the wild type, which were targeted for debugging. The effectiveness of the assay was demonstrated by phenotypic profiling of all intermediate synthetic strains of the synthetic yeast chromosome XII. Subsequently, the assay was applied during the process of constructing another synthetic chromosome. Furthermore, we designed an efficient chromosome assembly strategy that integrates iterative megachunk construction with CRISPR/Cas9-mediated assembly of synthetic segments. Together, the semiquantitative assay and refined assembly strategy could greatly facilitate synthetic genomics projects by improving efficiency during both debugging and construction.

A compact synthetic pathway rewires cancer signaling to therapeutic effector release.

An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9-mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Engineering the ribosomal DNA in a megabase synthetic chromosome.

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.