The combination of doxorubicin (Adriamycin) and cyclophosphamide, referred to as AC chemotherapy, is commonly used for the clinical treatment of breast and other cancers. Both agents target DNA with cyclophosphamide causing alkylation damage and doxorubicin stabilizing the topoisomerase II–DNA complex. We hypothesize a new mechanism of action whereby both agents work in concert. DNA alkylating agents, such as nitrogen mustards, increase the number of apurinic/apyrimidinic (AP) sites through deglycosylation of labile alkylated bases. Herein, we demonstrate that anthracyclines with aldehyde-reactive primary and secondary amines form covalent Schiff base adducts with AP sites in a 12-mer DNA duplex, calf thymus DNA, and MDA-MB-231 human breast cancer cells treated with nor-nitrogen mustard and the anthracycline mitoxantrone. The anthracycline–AP site conjugates are characterized and quantified by mass spectrometry after NaB(CN)H3 or NaBH4 reduction of the Schiff base. If stable, the anthracycline–AP site conjugates represent bulky adducts that may block DNA replication and contribute to the cytotoxic mechanism of therapies involving combinations of anthracyclines and DNA alkylating agents.

Non-enzymatic posttranslational modifications are believed to affect at least 30% of human proteins, commonly termed glycation. Many of these modifications are implicated in various pathological conditions, e.g., cataract, diabetes, neurodegenerative diseases, and cancer. Chemical protein synthesis enables access to full-length proteins carrying site-specific modifications. One such modification, argpyrimidine (Apy), has been detected in human small heat shock protein Hsp27 and closely related proteins in patient-derived tissues. Thus far, studies have looked into only artificial mixtures of Apy modifications, and only one has analyzed Apy188. We were interested in understanding the impact of such individual Apy modifications on five different arginine sites within the crucial N-terminal domain of Hsp27. By combining protein semisynthesis with biochemical assays on semisynthetic Hsp27 analogues with single-point Apy modification at those sites, we have shown how a seemingly minimal modification within this region results in dramatically altered functional attributes.

Boronic acid-containing fluorescent molecules have been widely used to sense hydrogen peroxide and peroxynitrite, which are important reactive oxygen and nitrogen species in biological systems. However, it has been challenging to gain specificity. Our previous studies developed genetically encoded, green fluorescent peroxynitrite biosensors by genetically incorporating a boronic acid-containing noncanonical amino acid (ncAA), p-boronophenylalanine (pBoF), into the chromophore of circularly permuted green fluorescent proteins (cpGFPs). In this work, we introduced pBoF to amino acid residues spatially close to the chromophore of an enhanced circularly permuted red fluorescent protein (ecpApple). Our effort has resulted in two responsive ecpApple mutants: one bestows reactivity toward both peroxynitrite and hydrogen peroxide, while the other, namely, pnRFP, is a selective red fluorescent peroxynitrite biosensor. We characterized pnRFP in vitro and in live mammalian cells. We further studied the structure and sensing mechanism of pnRFP using X-ray crystallography, 11B-NMR, and computational methods. The boron atom in pnRFP adopts an sp2-hybridization geometry in a hydrophobic pocket, and the reaction of pnRFP with peroxynitrite generates a product with a twisted chromophore, corroborating the observed “turn-off” fluorescence response. Thus, this study extends the color palette of genetically encoded peroxynitrite biosensors, provides insight into the response mechanism of the new biosensor, and demonstrates the versatility of using protein scaffolds to modulate chemoreactivity.

Desferrioxamine siderophores are assembled by the nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase enzyme DesD via ATP-dependent iterative condensation of three N1-hydroxy-N1-succinyl-cadaverine (HSC) units. Current knowledge of NIS enzymology and the desferrioxamine biosynthetic pathway does not account for the existence of most known members of this natural product family, which differ in substitution patterns of the N- and C-termini. The directionality of desferrioxamine biosynthetic assembly, N-to-C versus C-to-N, is a longstanding knowledge gap that is limiting further progress in understanding the origins of natural products in this structural family. Here, we establish the directionality of desferrioxamine biosynthesis using a chemoenzymatic approach with stable isotope incorporation and dimeric substrates. We propose a mechanism where DesD catalyzes the N-to-C condensation of HSC units to establish a unifying biosynthetic paradigm for desferrioxamine natural products in Streptomyces.

Three supplementary movies (Movies 1–3) mentioned in the original manuscript and its Supporting Information had inadvertently not been uploaded for publication. These movies are now being made available with this correction. The Supporting Information contains additional figures and tables with spectroscopic (UV–vis, NMR) and spectrometric (MS) data, genomic information, fluorescence microscopy images, associated data plots (in one PDF) as well as three supplementary movies. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.3c00388. Movie 1 (MP4) Movie 2 (MP4) Movie 3 (MP4) Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html. This article has not yet been cited by other publications. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.3c00388. Movie 1 (MP4) Movie 2 (MP4) Movie 3 (MP4) Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

The bacterium Escherichia coli possesses 16S and 23S rRNA strands that have 36 chemical modification sites with 17 different structures. Nanopore direct RNA sequencing using a protein nanopore sensor and helicase brake, which is also a sensor, was applied to the rRNAs. Nanopore current levels, base calling profile, and helicase dwell times for the modifications relative to unmodified synthetic rRNA controls found signatures for nearly all modifications. Signatures for clustered modifications were determined by selective sequencing of writer knockout E. coli and sequencing of synthetic RNAs utilizing some custom-synthesized nucleotide triphosphates for their preparation. The knowledge of each modification’s signature, apart from 5-methylcytidine, was used to determine how metabolic and cold-shock stress impact rRNA modifications. Metabolic stress resulted in either no change or a decrease, and one site increased in modification occupancy, while cold-shock stress led to either no change or a decrease. The double modification m4Cm1402 resides in 16S rRNA, and it decreased with both stressors. Using the helicase dwell time, it was determined that the N4 methyl group is lost during both stressors, and the 2′-OMe group remained. In the ribosome, this modification stabilizes binding to the mRNA codon at the P-site resulting in increased translational fidelity that is lost during stress. The E. coli genome has seven rRNA operons (rrn), and the earlier studies aligned the nanopore reads to a single operon (rrnA). Here, the reads were aligned to all seven operons to identify operon-specific changes in the 11 pseudouridines. This study demonstrates that direct sequencing for >16 different RNA modifications in a strand is achievable.

Modular polyketide synthases (PKSs) are attractive targets for the directed, biosynthetic production of platform chemicals and pharmaceuticals by protein engineering. In this study, we analyze docking domains from the 6-deoxyerythronolide B synthase, SYNZIP domains, and the SpyCatcher:SpyTag complex as engineering tools to couple the polypeptides VemG and VemH to functional venemycin synthases. Our data show that the high-affinity interaction or covalent connection of modules, enabled by SYNZIP domains and the SpyCatcher:SpyTag complex, can be advantageous, e.g., in synthesis at low protein concentrations, but their rigidity and steric demand decrease synthesis rates. However, we also show that efficiency can be recovered when inserting a hinge region distant from the rigid interface. This study demonstrates that engineering approaches should take the conformational properties of modular PKSs into account and establishes a three-polypeptide split venemycin synthase as an exquisite in vitro platform for the analysis and engineering of modular PKSs.

Herbicides in the widely used chloroacetanilide class harbor a potent electrophilic moiety, which can damage proteins through nucleophilic substitution. In general, damaged proteins are subject to misfolding. Accumulation of misfolded proteins compromises cellular integrity by disrupting cellular proteostasis networks, which can further destabilize the cellular proteome. While direct conjugation targets can be discovered through affinity-based protein profiling, there are few approaches to probe how cellular exposure to toxicants impacts the stability of the proteome. We apply a quantitative proteomics methodology to identify chloroacetanilide-destabilized proteins in HEK293T cells based on their binding to the H31Q mutant of the human Hsp40 chaperone DNAJB8. We find that a brief cellular exposure to the chloroacetanilides acetochlor, alachlor, and propachlor induces misfolding of dozens of cellular proteins. These herbicides feature distinct but overlapping profiles of protein destabilization, highly concentrated in proteins with reactive cysteine residues. Consistent with the recent literature from the pharmacology field, reactivity is driven by neither inherent nucleophilic nor electrophilic reactivity but is idiosyncratic. We discover that propachlor induces a general increase in protein aggregation and selectively targets GAPDH and PARK7, leading to a decrease in their cellular activities. Hsp40 affinity profiling identifies a majority of propachlor targets identified by competitive activity-based protein profiling (ABPP), but ABPP can only identify about 10% of protein targets identified by Hsp40 affinity profiling. GAPDH is primarily modified by the direct conjugation of propachlor at a catalytic cysteine residue, leading to global destabilization of the protein. The Hsp40 affinity strategy is an effective technique to profile cellular proteins that are destabilized by cellular toxin exposure. Raw proteomics data is available through the PRIDE Archive at PXD030635.

PROteolysis TArgeting Chimeras (PROTACs) are of significant current interest for the development of probe molecules and drug leads. However, they suffer from certain limitations. PROTACs are rule-breaking molecules with sub-optimal cellular permeability, solubility, and other drug-like properties. In particular, they exhibit an unusual dose–response curve where high concentrations of the bivalent molecule inhibit degradation activity, a phenomenon known as the hook effect. This will likely complicate their use in vivo. In this study, we explore a novel approach to create PROTACs that do not exhibit a hook effect. This is achieved by equipping the target protein and E3 ubiquitin ligase ligands with functionalities that undergo rapid and reversible covalent assembly in cellulo. We report the development of Self-Assembled Proteolysis Targeting Chimeras that mediate the degradation of the Von Hippel–Lindau E3 ubiquitin ligase and do not evince a hook effect.

Protein-based therapeutics, such as monoclonal antibodies and cytokines, are important therapies for various pathophysiological conditions such as oncology, autoimmune disorders, and viral infections. However, the wide application of such protein therapeutics is often hindered by dose-limiting toxicities and adverse effects, namely, cytokine storm syndrome, organ failure, and others. Therefore, spatiotemporal control of the activities of these proteins is crucial to further expand their application. Here, we report the design and application of small-molecule-controlled switchable protein therapeutics by taking advantage of a previously engineered OFF-switch system. We used the Rosetta modeling suite to computationally optimize the affinity between B-cell lymphoma 2 (Bcl-2) protein and a previously developed computationally designed protein partner (LD3) to obtain a fast and efficient heterodimer disruption upon the addition of a competing drug (Venetoclax). The incorporation of the engineered OFF-switch system into anti-CTLA4, anti-HER2 antibodies, or an Fc-fused IL-15 cytokine demonstrated an efficient disruption in vitro, as well as fast clearance in vivo upon the addition of the competing drug Venetoclax. These results provide a proof-of-concept for the rational design of controllable biologics by introducing a drug-induced OFF-switch into existing protein-based therapeutics.

The emergence of multidrug-resistant pathogens poses a threat to public health and requires new antimicrobial agents. As the archetypal glycopeptide antibiotic (GPA) used against drug-resistant Gram-positive pathogens, vancomycin provides a promising starting point. Peripheral alterations to the vancomycin scaffold have enabled the development of new GPAs. However, modifying the core remains challenging due to the size and complexity of this compound family. The recent successful chemoenzymatic synthesis of vancomycin suggests that such an approach can be broadly applied. Herein, we describe the expansion of chemoenzymatic strategies to encompass type II GPAs bearing all aromatic amino acids through the production of the aglycone analogue of keratinimicin A, a GPA that is 5-fold more potent than vancomycin against Clostridioides difficile. In the course of these studies, we found that the cytochrome P450 enzyme OxyBker boasts both broad substrate tolerance and remarkable selectivity in the formation of the first aryl ether cross-link on the linear peptide precursors. The X-ray crystal structure of OxyBker, determined to 2.8 Å, points to structural features that may contribute to these properties. Our results set the stage for using OxyBker broadly as a biocatalyst toward the chemoenzymatic synthesis of diverse GPA analogues.

Targeted protein degradation is an emerging technology that can be used for modulating the activity of epigenetic protein targets. Among bromodomain-containing proteins, a number of degraders for the BET family have been developed, while non-BET bromodomains remain underexplored. Several of these proteins are subunits in chromatin remodeling complexes often associated with oncogenic roles. Here, we describe the design of class I (BPTF and CECR2) and IV (BRD9) bromodomain-targeting degraders based on two scaffolds derived from pyridazinone and pyrimidine-based heterocycles. We evaluate various exit vectors and linkers to identify analogues that demonstrate selectivity within these families. We further use an in-cell NanoBRET assay to demonstrate that these heterobifunctional molecules are cell-permeable, form ternary complexes, and can degrade nanoluciferase–bromodomain fusions. As a first example of a CECR2 degrader, we observe that our pyrimidine-based analogues degrade endogenous CECR2 while showing a smaller effect on BPTF levels. The pyridazinone-based compounds did not degrade BPTF when observed through Western blotting, further supporting a more challenging target for degradation and a goal for future optimization.

It is of great interest to probe glycosylation in primary neuron cultures. However, per-O-acetylated clickable unnatural sugars, which have been routinely utilized in metabolic glycan labeling (MGL) for analyzing glycans, showed cytotoxicity to cultured primary neurons and thus led to the speculation that MGL was not compatible with primary neuron cell cultures. Here, we uncovered that neuron cytotoxicity of per-O-acetylated unnatural sugars was related to their reactions with protein cysteines via non-enzymatic S-glyco-modification. The modified proteins were enriched in biological functions related to microtubule cytoskeleton organization, positive regulation of axon extension, neuron projection development, and axonogenesis. We thus established MGL in cultured primary neurons without cytotoxicity using S-glyco-modification-free unnatural sugars including ManNAz, 1,3-Pr2ManNAz, and 1,6-Pr2ManNAz, which allowed for visualization of cell-surface sialylated glycans, probing the dynamics of sialylation, and large-scale identification of sialylated N-linked glycoproteins and the modification sites in primary neurons. Particularly, a total of 505 sialylated N-glycosylation sites distributed on 345 glycoproteins were identified by 1,6-Pr2ManNAz.

Styrene dyes are useful imaging probes and fluorescent sensors due to their strong fluorogenic responses to environmental changes or binding macromolecules. Previously, indole-containing styrene dyes have been reported to selectively bind RNA in the nucleolus and cytoplasm. However, the application of these indole-based dyes in cell imaging is limited by their moderate fluorescence enhancement and quantum yields, as well as relatively high background associated with these green-emitting dyes. In this work, we have investigated the positional and electronic effects of the electron donor by generating regioisomeric and isosteric analogues of the indole ring. Select probes exhibited large Stokes shifts, enhanced molar extinction coefficients, and bathochromic shifts in their absorption and fluorescence wavelengths. In particular, the indolizine analogues displayed high membrane permeability, strong fluorogenic responses upon binding RNA, compatibility with fluorescence lifetime imaging microscopy (FLIM), low cytotoxicity, and excellent photostability. These indolizine dyes not only give rise to rapid, sensitive, and intense staining of nucleoli in live cells but can also resolve subnucleolar structures enabling highly detailed studies of nucleolar morphology. Furthermore, our dyes can partition into RNA coacervates and resolve the formation of multiphase complex coacervate droplets. These indolizine-containing styrene probes offer the highest fluorescence enhancement among the RNA-selective dyes reported in the literature; thus, these new dyes are excellent alternatives to the commercially available RNA dye, SYTO RNASelect, for visualizing RNA in live cells and in vitro.

Mammalian DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B, are key DNA methylation enzymes and play important roles in gene expression regulation. Dysregulation of DNMTs is linked to various diseases and carcinogenesis, and therefore except for the two approved anticancer azanucleoside drugs, various non-nucleoside DNMT inhibitors have been identified and reported. However, the underlying mechanisms for the inhibitory activity of these non-nucleoside inhibitors still remain largely unknown. Here, we systematically tested and compared the inhibition activities of five non-nucleoside inhibitors toward the three human DNMTs. We found that harmine and nanaomycin A blocked the methyltransferase activity of DNMT3A and DNMT3B more efficiently than resveratrol, EGCG, and RG108. We further determined the crystal structure of harmine in complex with the catalytic domain of the DNMT3B-DNMT3L tetramer revealing that harmine binds at the adenine cavity of the SAM-binding pocket in DNMT3B. Our kinetics assays confirm that harmine competes with SAM to competitively inhibit DNMT3B-3L activity with a Ki of 6.6 μM. Cell-based studies further show that harmine treatment inhibits castration-resistant prostate cancer cell (CRPC) proliferation with an IC50 of ∼14 μM. The CPRC cells treated with harmine resulted in reactivating silenced hypermethylated genes compared to the untreated cells, and harmine cooperated with an androgen antagonist, bicalutamide, to effectively inhibit the proliferation of CRPC cells. Our study thus reveals, for the first time, the inhibitory mechanism of harmine on DNMTs and highlights new strategies for developing novel DNMT inhibitors for cancer treatment.

Glycosaminoglycan synthases have immense potential in applications involving synthesis of oligosaccharides, using enzymatic approaches and construction of cell factories that produce polysaccharides as critical metabolic components. However, the use of high-throughput activity assays to screen for the evolution of these enzymes can be challenging because there are no significant changes in fluorescence or absorbance associated with glycosidic bond formation. Here, using incorporation of azido-labeled N-acetylhexosamine analogs into bacterial capsule polysaccharides via bacterial metabolism and bioorthogonal chemistry, fluorophores were specifically introduced onto cell surfaces. Furthermore, correlations between detectable fluorescence signals and the polysaccharide-synthesizing capacity of individual bacteria were established. Among 10 candidate genes, 6 members of the chondroitin synthase family were quickly identified in a recombinant Bacillus subtilis host strain. Additionally, directed evolution of heparosan synthase was successfully performed using fluorescence-activated cell sorting of recombinant Escherichia coli O10:K5(L):H4, yielding several mutants with increased activity. Cell-based approaches that selectively detect the presence or absence of synthases within an individual colony of bacterial cells, as well as their level of activity, have broad potential in the exploration and engineering of glycosaminoglycan synthases. These approaches also support the creation of novel strategies for high-throughput screening of enzyme activity based on cell systems.

Ribonucleases (RNases) cleave and process RNAs, thereby regulating the biogenesis, metabolism, and degradation of coding and noncoding RNAs. Thus, small molecules targeting RNases have the potential to perturb RNA biology, and RNases have been studied as therapeutic targets of antibiotics, antivirals, and agents for autoimmune diseases and cancers. Additionally, the recent advances in chemically induced proximity approaches have led to the discovery of bifunctional molecules that target RNases to achieve RNA degradation or inhibit RNA processing. Here, we summarize the efforts that have been made to discover small-molecule inhibitors and activators targeting bacterial, viral, and human RNases. We also highlight the emerging examples of RNase-targeting bifunctional molecules and discuss the trends in developing such molecules for both biological and therapeutic applications.

The constant and the sudden emergence of zoonotic human and animal viruses is a significant threat to human health, the world economy, and the world food supply. This has necessitated the development of broad-spectrum therapeutic strategies to combat these emerging pathogens. Mechanisms that are essential for viral replication and propagation have been successfully targeted in the past to develop broad-spectrum therapeutics that can be readily repurposed to combat new zoonotic pathogens. Because of the importance of viral RNA capping enzymes to viral replication and pathogenesis, as well as their presence in both DNA and RNA viruses, these viral proteins have been a long-standing therapeutic target. Here, we use genome sequencing information and yeast-based platforms (YeRC0M) to identify, characterize, and target viral genome-encoded essential RNA capping enzymes from emerging strains of DNA viruses, i.e., Monkeypox virus and African Swine Fever Virus, which are a significant threat to human and domestic animal health. We first identified and biochemically characterized these viral RNA capping enzymes and their necessary protein domains. We observed significant differences in functional protein domains and organization for RNA capping enzymes from emerging DNA viruses in comparison to emerging RNA viruses. We also observed several differences in the biochemical properties of these viral RNA capping enzymes using our phenotypic yeast-based approaches (YeRC0M) as compared to the previous in vitro studies. Further, using directed evolution, we were able to identify inactivation and attenuation mutations in these essential viral RNA capping enzymes; these data could have implications on virus biocontainment as well as live attenuated vaccine development. We also developed methods that would facilitate high-throughput phenotypic screening to identify broad-spectrum inhibitors that selectively target viral RNA capping enzymes over host RNA capping enzymes. As demonstrated here, our approaches to identify, characterize, and target viral genome-encoded essential RNA capping enzymes are highly modular and can be readily adapted for targeting emerging viral pathogens as well as their variants that emerge in the future.

Abberent protein–protein interactions (aPPIs) are associated with an array of pathological conditions, which make them important therapeutic targets. The aPPIs are mediated via specific chemical interactions that spread over a large and hydrophobic surface. Therefore, ligands that can complement the surface topography and chemical fingerprints could manipulate aPPIs. Oligopyridylamides (OPs) are synthetic protein mimetics that have been shown to manipulate aPPIs. However, the previous OP library used to disrupt these aPPIs was moderate in number (∼30 OPs) with very limited chemical diversity. The onus is on the laborious and time-consuming synthetic pathways with multiple chromatography steps. We have developed a novel chromatography-free technique to synthesize a highly diverse chemical library of OPs using a “common-precursor” approach. We significantly expanded the chemical diversity of OPs using a chromatography-free high-yielding method. To validate our novel approach, we have synthesized an OP with identical chemical diversity to a pre-existing OP-based potent inhibitor of Aβ aggregation, a process central to Alzheimer’s disease (AD). The newly synthesized OP ligand (RD242) was very potent in inhibiting Aβ aggregation and rescuing AD phenotypes in an in vivo model. Moreover, RD242 was very effective in rescuing AD phenotypes in a post-disease onset AD model. We envision that our “common-precursor” synthetic approach will have tremendous potential as it is expandable for other oligoamide scaffolds to enhance affinity for disease-relevant targets.

“Reagentless” immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.