Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Ras-mutant cancers are sensitive to small molecule inhibition of V-type ATPases in mice.

Mutations in Ras family proteins are implicated in 33% of human cancers, but direct pharmacological inhibition of Ras mutants remains challenging. As an alternative to direct inhibition, we screened for sensitivities in Ras-mutant cells and discovered 249C as a Ras-mutant selective cytotoxic agent with nanomolar potency against a spectrum of Ras-mutant cancers. 249C binds to vacuolar (V)-ATPase with nanomolar affinity and inhibits its activity, preventing lysosomal acidification and inhibiting autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. Unexpectedly, potency of 249C varies with the identity of the Ras driver mutation, with the highest potency for KRASG13D and G12V both in vitro and in vivo, highlighting a mutant-specific dependence on macropinocytosis and lysosomal pH. Indeed, 249C potently inhibits tumor growth without adverse side effects in mouse xenografts of KRAS-driven lung and colon cancers. A comparison of isogenic SW48 xenografts with different KRAS mutations confirmed that KRASG13D/+ (followed by G12V/+) mutations are especially sensitive to 249C treatment. These data establish proof-of-concept for targeting V-ATPase in cancers driven by specific KRAS mutations such as KRASG13D and G12V.

Leaks in the Pipeline: a Failure Analysis of Gram-Negative Antibiotic Development from 2010 to 2020.

The World Health Organization (WHO) has warned that our current arsenal of antibiotics is not innovative enough to face impending infectious diseases, especially those caused by multidrug-resistant Gram-negative pathogens. Although the current preclinical pipeline is well stocked with novel candidates, the last U.S. Food and Drug Administration (FDA)-approved antibiotic with a novel mechanism of action against Gram-negative bacteria was discovered nearly 60 years ago. Of all the antibiotic candidates that initiated investigational new drug (IND) applications in the 2000s, 17% earned FDA approval within 12 years, while an overwhelming 62% were discontinued in that time frame. These "leaks" in the clinical pipeline, where compounds with clinical potential are abandoned during clinical development, indicate that scientific innovations are not reaching the clinic and providing benefits to patients. This is true for not only novel candidates but also candidates from existing antibiotic classes with clinically validated targets. By identifying the sources of the leaks in the clinical pipeline, future developmental efforts can be directed toward strategies that are more likely to flow into clinical use. In this review, we conduct a detailed failure analysis of clinical candidates with Gram-negative activity that have fallen out of the clinical pipeline over the past decade. Although limited by incomplete data disclosure from companies engaging in antibiotic development, we attempt to distill the developmental challenges faced by each discontinued candidate. It is our hope that this insight can help de-risk antibiotic development and bring new, effective antibiotics to the clinic.

Roadmap for Optimizing and Broadening Antibody-Based PROTACs for Degradation of Cell Surface Proteins.

Targeted protein degradation is a promising therapeutic strategy capable of overcoming the limitations of traditional occupancy-based inhibitors. By ablating all of the associated functions of a protein at once, the event-driven pharmacology of degrader technologies has recently enabled the targeting of proteins that have been historically deemed "undruggable". Most degradation strategies utilize the ubiquitin-proteasome system to mediate intracellular target degradation and are thus limited to targeting proteins with cytoplasmic domains. While some of these strategies, such as PROTACs, have shown great promise, there is a need for new modalities that can be applied to specifically target cell surface proteins. We previously described the development of an antibody-based PROTAC (AbTAC) that utilizes genetically encoded IgG bispecific antibody scaffolds to bring the cell surface E3-ligase RNF43 into the proximity of a membrane protein of interest (POI) to mediate its degradation. Here, we employ rational protein engineering strategies to interrogate and optimize the properties necessary for efficient degradation of two therapeutically important membrane proteins, PD-L1 and EGFR. We develop multiple antibodies to RNF43 and show that the specific antibody binding epitopes on RNF43 and the POI are more important than the affinities of the AbTAC antibodies. We further expand the available repertoire of E3 ligases by co-opting the E3-ligase ZNRF3 to degrade both PD-L1 and EGFR and show similar importance of epitope for degradation efficiency. Importantly, we show that both RNF43 and ZNRF3 AbTACs do not potentiate unwanted WNT signaling. Lastly, we find that these AbTACs can be even further improved by exploring various dual-binding and IgG scaffolds that range in flexibility, valency, and orientation of the binding arms. These structure-activity and mechanistic studies provide a roadmap for optimizing the development of AbTACs, thereby greatly expanding their utility for targeted cell surface protein degradation.

Biotin as a Reactive Handle to Selectively Label Proteins and DNA with Small Molecules.

Biotin is a common functional handle for bioconjugation to proteins and DNA, but its uses are limited to protein-containing conjugation partners such as streptavidin and derivatives thereof. Recently, oxaziridine reagents were developed that selectively conjugate the thioether of methionines on the surface of proteins, a method termed redox-activated chemical tagging (ReACT). These reagents generate sulfimide linkages that range in stability depending on the solvent accessibility and substitutions on the oxaziridine. Here we show that oxaziridine reagents react rapidly with the thioether in biotin to produce sulfimide products that are stable for more than 10 d at 37 °C. This method, which we call biotin redox-activated chemical tagging (BioReACT), expands the utility of biotin labeling and enables a predictable and stable chemical conjugation to biomolecules without the need to screen for a suitable methionine conjugation site. We demonstrate the versatility of this approach by producing a fluorescently labeled antibody, an antibody-drug conjugate, and a small molecule-conjugated oligonucleotide. We anticipate that BioReACT will be useful to rapidly introduce biorthogonal handles into biomolecules using biotin, a functional group that is widespread and straightforward to install.

Examining Gender Imbalance in Chemistry Authorship.

Despite decades of progress toward a more equitable society, gender representation in the sciences continues to be heavily skewed toward men. We were interested in gender representation in chemistry through the lens of scientific publishing. Publications are a central academic currency and are critical for funding, recruiting, and promotion in academia. Here we report the results of an analysis that compared the percentage of female first and last authors across 10 chemistry, 3 chemical biology, and 3 general journals from 2005 to 2020. We show that women are substantially underrepresented in chemistry authorship even when compared with their relative populations in academia and are not predicted to achieve parity within the next 50 years at the current rate in any journal. Our findings highlight the need for changes to the publishing process to achieve a more equitable publishing environment.

Label-retention expansion microscopy.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

Modular synthesis of streptogramin antibiotics.

Streptogramins are antibiotics produced by several species of Streptomyces bacteria that are used in both human and veterinary medicine. Group A streptogramins comprise 23-membered macrocyclic polyketide/nonribosomal peptide hybrids for which several innovative, fully synthetic routes have been developed. Herein we describe in detail our scalable routes to natural group A streptogramins and compare these routes to other reported syntheses.

Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division.

In the developing embryos, the cortical polarity regulator Par-3 is critical for establishing Notch signaling asymmetry between daughter cells during asymmetric cell division (ACD). How cortically localized Par-3 establishes asymmetric Notch activity in the nucleus is not understood. Here, using in vivo time-lapse imaging of mitotic radial glia progenitors in the developing zebrafish forebrain, we uncover that during horizontal ACD along the anteroposterior embryonic axis, endosomes containing the Notch ligand DeltaD (Dld) move toward the cleavage plane and preferentially segregate into the posterior (subsequently basal) Notchhi daughter. This asymmetric segregation requires the activity of Par-3 and dynein motor complex. Using label retention expansion microscopy, we further detect Par-3 in the cytosol colocalizing the dynein light intermediate chain 1 (Dlic1) onto Dld endosomes. Par-3, Dlic1, and Dld are associated in protein complexes in vivo. Our data reveal an unanticipated mechanism by which cytoplasmic Par-3 directly polarizes Notch signaling components during ACD.

Modular Chemical Synthesis of Streptogramin and Lankacidin Antibiotics.

Continued, rapid development of antimicrobial resistance has become worldwide health crisis and a burden on the global economy. Decisive and comprehensive action is required to slow down the spread of antibiotic resistance, including increased investment in antibiotic discovery, sustainable policies that provide returns on investment for newly launched antibiotics, and public education to reduce the overusage of antibiotics, especially in livestock and agriculture. Without significant changes in the current antibiotic pipeline, we are in danger of entering a post-antibiotic era.In this Account, we summarize our recent efforts to develop next-generation streptogramin and lankacidin antibiotics that overcome bacterial resistance by means of modular chemical synthesis. First, we describe our highly modular, scalable route to four natural group A streptogramins antibiotics in 6-8 steps from seven simple chemical building blocks. We next describe the application of this route to the synthesis of a novel library of streptogramin antibiotics informed by in vitro and in vivo biological evaluation and high-resolution cryo-electron microscopy. One lead compound showed excellent inhibitory activity in vitro and in vivo against a longstanding streptogramin-resistance mechanism, virginiamycin acetyltransferase. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.Second, we recount our modular approaches toward lankacidin antibiotics. Lankacidins are a group of polyketide natural products with activity against several strains of Gram-positive bacteria but have not been deployed as therapeutics due to their chemical instability. We describe a route to several diastereomers of 2,18-seco-lankacidinol B in a linear sequence of ≤8 steps from simple building blocks, resulting in a revision of the C4 stereochemistry. We next detail our modular synthesis of several diastereoisomers of iso-lankacidinol that resulted in the structural reassignment of this natural product. These structural revisions raise interesting questions about the biosynthetic origin of lankacidins, all of which possessed uniform stereochemistry prior to these findings. Finally, we summarize the ability of several iso- and seco-lankacidins to inhibit the growth of bacteria and to inhibit translation in vitro, providing important insights into structure-function relationships for the class.

Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1.

Targeted protein degradation has emerged as a new paradigm to manipulate cellular proteostasis. Proteolysis-targeting chimeras (PROTACs) are bifunctional small molecules that recruit an E3 ligase to a target protein of interest, promoting its ubiquitination and subsequent degradation. Here, we report the development of antibody-based PROTACs (AbTACs), fully recombinant bispecific antibodies that recruit membrane-bound E3 ligases for the degradation of cell-surface proteins. We show that an AbTAC can induce the lysosomal degradation of programmed death-ligand 1 by recruitment of the membrane-bound E3 ligase RNF43. AbTACs represent a new archetype within the PROTAC field to target cell-surface proteins with fully recombinant biological molecules.

Platform to Discover Protease-Activated Antibiotics and Application to Siderophore-Antibiotic Conjugates.

Here we present a platform for discovery of protease-activated prodrugs and apply it to antibiotics that target Gram-negative bacteria. Because cleavable linkers for prodrugs had not been developed for bacterial proteases, we used substrate phage to discover substrates for proteases found in the bacterial periplasm. Rather than focusing on a single protease, we used a periplasmic extract of E. coli to find sequences with the greatest susceptibility to the endogenous mixture of periplasmic proteases. Using a fluorescence assay, candidate sequences were evaluated to identify substrates that release native amine-containing payloads. We next designed conjugates consisting of (1) an N-terminal siderophore to facilitate uptake, (2) a protease-cleavable linker, and (3) an amine-containing antibiotic. Using this strategy, we converted daptomycin-which by itself is active only against Gram-positive bacteria-into an antibiotic capable of targeting Gram-negative Acinetobacter species. We similarly demonstrated siderophore-facilitated delivery of oxazolidinone and macrolide antibiotics into a number of Gram-negative species. These results illustrate this platform's utility for development of protease-activated prodrugs, including Trojan horse antibiotics.

Dual antagonists of α5ß1/αvß1 integrin for airway hyperresponsiveness.

Inhibition of integrin α5ß1 emerges as a novel therapeutic option to block transmission of contractile forces during asthma attack. We designed and synthesized novel inhibitors of integrin α5ß1 by backbone replacement of known αvß1 integrin inhibitors. These integrin α5ß1 inhibitors also retain the nanomolar potency against αvß1 integrin, which shows promise for developing dual integrin α5ß1/αvß1 inhibitor. Introduction of hydrophobic adamantane group significantly boosted the potency as well as selectivity over integrin αvß3. We also demonstrated one of the inhibitors (11) reduced airway hyperresponsiveness in ex vivo mouse tracheal ring assay. Results from this study will help guide further development of integrin α5ß1 inhibitors as potential novel asthma therapeutics.

Synthetic group A streptogramin antibiotics that overcome Vat resistance.

Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Modular Approaches to Lankacidin Antibiotics.

Lankacidins are a class of polyketide natural products isolated from Streptomyces spp. that show promising antimicrobial activity. Owing to their complex molecular architectures and chemical instability, structural assignment and derivatization of lankacidins are challenging tasks. Herein we describe three fully synthetic approaches to lankacidins that enable access to new structural variability within the class. We use these routes to systematically generate stereochemical derivatives of both cyclic and acyclic lankacidins. Additionally, we access a new series of lankacidins bearing a methyl group at the C4 position, a modification intended to increase chemical stability. In the course of this work, we discovered that the reported structures for two natural products of the lankacidin class were incorrect, and we determine the correct structures of 2,18-seco-lankacidinol B and iso-lankacidinol. We also evaluate the ability of several iso- and seco-lankacidins to inhibit the growth of bacteria and to inhibit translation in vitro. This work grants insight into the rich chemical complexity of this class of antibiotics and provides an avenue for further structural derivatization.

Photocaged Quinone Methide Crosslinkers for Light-Controlled Chemical Crosslinking of Protein-Protein and Protein-DNA Complexes.

Small-molecule crosslinkers are invaluable for probing biomolecular interactions and for crosslinking mass spectrometry. Existing chemical crosslinkers target only a small selection of amino acids, while conventional photo-crosslinkers target almost all residues non-specifically, complicating data analysis. Herein, we report photocaged quinone methide (PQM)-based crosslinkers that target nine nucleophilic residues through Michael addition, including Gln, Arg, and Asn, which are inaccessible to existing chemical crosslinkers. PQM crosslinkers were used in vitro, in Escherichia coli, and in mammalian cells to crosslink dimeric proteins and endogenous membrane receptors. The heterobifunctional crosslinker NHQM could crosslink proteins to DNA, for which few crosslinkers exist. The photoactivatable reactivity of these crosslinkers and their ability to target multiple amino acids will enhance the use of chemical crosslinking for studies of protein-protein and protein-DNA networks and for structural biology.

A concise route to virginiamycin M2.

Modular, fully synthetic routes to structurally complex natural products provide useful avenues to access chemical diversity. Herein we report a concise route to virginiamycin M2, a member of the group A streptogramin class of natural products that inhibits bacterial protein synthesis. Our approach features a longest linear sequence of six steps from 7 simple building blocks, and is the shortest and highest yielding synthesis of any member of the streptogramin class reported to date. We believe this route will enable access to unexplored structural diversity and may serve as a useful tool to improve the therapeutic potential of the streptogramin class of antibiotics.

Synthesis, Structural Reassignment, and Antibacterial Evaluation of 2,18-Seco-Lankacidinol†B.

Lankacidins are a group of polyketide natural products with activity against several strains of Gram-positive bacteria. We developed a route to stereochemically diverse variants of 2,18-seco-lankacidinol B and found that the stereochemical assignment at C4 requires revision. This has interesting implications for the biosynthesis of natural products of the lankacidin class, all of which possessed uniform stereochemistry prior to this finding. We have evaluated 2,18-seco-lankacidinol B and three stereochemical derivatives against a panel of pathogenic Gram-positive and Gram-negative bacteria.

Modular, Scalable Synthesis of Group A Streptogramin Antibiotics.

Streptogramin antibiotics are used clinically to treat multidrug-resistant bacterial infections, but their poor physicochemical properties and narrow spectra of activity have limited their utility. New methods to chemically modify streptogramins would enable structural optimization to overcome these limitations as well as to combat growing resistance to the class. Here we report a modular, scalable synthesis of group A streptogramin antibiotics that proceeds in 6-8 linear steps from simple chemical building blocks. We have applied our route to the synthesis of four natural products in this class including two that have never before been accessed by fully synthetic routes. We anticipate that this work will lead to the discovery of new streptogramin antibiotics that overcome previous limitations of the class.

A platform for the discovery of new macrolide antibiotics.

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.