Biocatalytic Lactone Carbene C–H, B–H, and N–H Insertion Reactions Enabled by Directed Evolution

Enzymes are biological catalysts, and they accelerate reactions by lowering the activation barrier. In nature, enzymes have been optimized by natural selection and possess precise three-dimensional active sites. With these active sites, they can typically catalyze reactions with high efficiency and specificity. Compared to traditional catalysts, enzymes are generally more environmentally friendly, and they can catalyze reactions in water and at ambient temperature and pressure. However, native enzymes are usually only well suited for a restricted range of substrates and are limited in the types of reactions they perform. The Arnold lab has recently focused on endowing enzymes with the ability to catalyze new-to-nature reactions through directed evolution. Here, we present a set of enzymes engineered for the ability to insert a lactone carbene into B–H, C–H, and N–H bonds with high yield and enantioselectivity. B–H insertion is achieved by engineered cytochrome c enzymes, while N–H and C–H insertions are achieved by engineered cytochrome P450 enzymes. With this work, we expand nature’s toolbox for lactone insertion chemistry. Since lactones are highly bioactive, these engineered enzymes could be powerful tools in the synthesis of a range of pharmaceuticals and natural product targets

Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion

Previous work has demonstrated that variants of a heme protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abiological carbene boron–hydrogen (B–H) bond insertion with high efficiency and selectivity. Here we investigated this carbon–boron bond-forming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BOR^(LAC) that shows high selectivity and efficiency for B–H insertion of 5- and 6-membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a highly twisted geometry of the 7-membered lactone carbene intermediate relative to 5- and 6-membered ones. Directed evolution of cytochrome c together with computational characterization of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H insertion to produce new lactone-based organoborons

Self-Assembled STING-Activating Coordination Nanoparticles for Cancer Immunotherapy and Vaccine Applications

The cGAS-STING pathway plays a crucial role in innate immune activation against cancer and infections, and STING agonists based on cyclic dinucleotides (CDN) have garnered attention for their potential use in cancer immunotherapy and vaccines. However, the limited drug-like properties of CDN necessitate an efficient delivery system to the immune system. To address these challenges, we developed an immunostimulatory delivery system for STING agonists. Here, we have examined aqueous coordination interactions between CDN and metal ions and report that CDN mixed with Zn2+ and Mn2+ formed distinctive crystal structures. Further pharmaceutical engineering led to the development of a functional coordination nanoparticle, termed the Zinc–Mn–CDN Particle (ZMCP), produced by a simple aqueous one-pot synthesis. Local or systemic administration of ZMCP exerted robust antitumor efficacy in mice. Importantly, recombinant protein antigens from SARS-CoV-2 can be simply loaded during the aqueous one-pot synthesis. The resulting ZMCP antigens elicited strong cellular and humoral immune responses that neutralized SARS-CoV-2, highlighting ZMCP as a self-adjuvant vaccine platform against COVID-19 and other infectious pathogens. Overall, this work establishes a paradigm for developing translational coordination nanomedicine based on drug–metal ion coordination and broadens the applicability of coordination medicine for the delivery of proteins and other biologics

Kinetics of diffusion-controlled enzymatic reactions with charged substrates

The Debye-Hückel limiting law (DHL) has often been used to estimate rate constants of diffusion-controlled reactions under different ionic strengths. Two main approximations are adopted in DHL: one is that the solution of the linearized Poisson-Boltzmann equation for a spherical cavity is used to estimate the excess electrostatic free energy of a solution; the other is that details of electrostatic interactions of the solutes are neglected. This makes DHL applicable only at low ionic strengths and dilute solutions (very low substrate/solute concentrations). We show in this work that through numerical solution of the Poisson-Nernst-Planck equations, diffusion-reaction processes can be studied at a variety of conditions including realistically concentrated solutions, high ionic strength, and certainly with non-equilibrium charge distributions. Reaction rate coefficients for the acetylcholine-acetylcholinesterase system are predicted to strongly depend on both ionic strength and substrate concentration. In particular, they increase considerably with increase of substrate concentrations at a fixed ionic strength, which is open to experimental testing. This phenomenon is also verified on a simple model, and is expected to be general for electrostatically attracting enzyme-substrate systems

Engineered Cytochrome c-Catalyzed Lactone-Carbene B–H Insertion

Previous work has demonstrated that variants of a heme protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abiological carbene boron–hydrogen (B–H) bond insertion with high efficiency and selectivity. Here we investigated this carbon–boron bond-forming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BOR^(LAC) that shows high selectivity and efficiency for B–H insertion of 5- and 6-membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a highly twisted geometry of the 7-membered lactone carbene intermediate relative to 5- and 6-membered ones. Directed evolution of cytochrome c together with computational characterization of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H insertion to produce new lactone-based organoborons

Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer.

Although targeted therapies often elicit profound initial patient responses, these effects are transient due to residual disease leading to acquired resistance. How tumors transition between drug responsiveness, tolerance and resistance, especially in the absence of preexisting subclones, remains unclear. In epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells, we demonstrate that residual disease and acquired resistance in response to EGFR inhibitors requires Aurora kinase A (AURKA) activity. Nongenetic resistance through the activation of AURKA by its coactivator TPX2 emerges in response to chronic EGFR inhibition where it mitigates drug-induced apoptosis. Aurora kinase inhibitors suppress this adaptive survival program, increasing the magnitude and duration of EGFR inhibitor response in preclinical models. Treatment-induced activation of AURKA is associated with resistance to EGFR inhibitors in vitro, in vivo and in most individuals with EGFR-mutant lung adenocarcinoma. These findings delineate a molecular path whereby drug resistance emerges from drug-tolerant cells and unveils a synthetic lethal strategy for enhancing responses to EGFR inhibitors by suppressing AURKA-driven residual disease and acquired resistance

Attribution of extreme precipitation in the lower reaches of the Yangtze River during May 2016

May 2016 was the third wettest May on record since 1961 over central eastern China based on station observations, with total monthly rainfall 40% more than the climatological mean for 1961–2013. Accompanying disasters such as waterlogging, landslides and debris flow struck part of the lower reaches of the Yangtze River. Causal influence of anthropogenic forcings on this event is investigated using the newly updated Met Office Hadley Centre system for attribution of extreme weather and climate events. Results indicate that there is a significant increase in May 2016 rainfall in model simulations relative to the climatological period, but this increase is largely attributable to natural variability. El Ni ̃no years have been found to be correlatedwith extreme rainfall in the Yangtze River region in previous studies—the strong El Ni ̃no of 2015–2016 may account for the extreme precipitation event in 2016. However, on smaller spatial scales we find that anthropogenic forcing has likely played a role in increasing the risk of extreme rainfall to the north of the Yangtze and decreasing it to the south

Nature of the Paleo-Pacific Subduction along the East Asian Continental Margin in the Mesozoic: insights from the sedimentary record of West Sarawak, Borneo

The Mesozoic subduction history of the Paleo-Pacific plate below the East Asian margin remains contentious, in part because the southern part is poorly understood. To address this, we conducted a sediment provenance study to constrain Mesozoic subduction history below West Sarawak, Borneo. A combination of detrital zircon U-Pb geochronology, heavy minerals, trace element, and bulk rock Nd isotope data were used to identify the tectonic events. The overall maturity of mineral assemblages, dominantly felsic sources, abundant Precambrian-aged zircons, and low εNd(0) values (average −13.07) seen in Late Triassic sedimentary rocks suggest a period of inactive subduction near Borneo. Slab shallowing subduction occurred between 200 and 170 Ma based on subdued magmatism and tectonic compression across West Sarawak. From c. 170 to 70 Ma there was widespread magmatism and we interpret the Paleo-Pacific slab steepened. Collectively, we show the Paleo-Pacific plate subduction had variable slab dip histories in Borneo