Combining recombinase polymerase amplification and DNA-templated reaction for SARS-CoV-2 sensing with dual fluorescence and lateral flow assay output.

The early phase of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was exacerbated by a diagnostic challenge of unprecedented magnitude. In the absence of effective therapeutics or vaccines, breaking the chain of transmission through early disease detection and patient isolation was the only means to control the growing pandemic. While polymerase chain reaction (PCR)-based methods and rapid-antigen tests rose to the occasion, the analytical challenge of rapid and sequence-specific nucleic acid-sensing at a point-of-care or home setting stimulated intense developments. Herein we report a method that combines recombinase polymerase amplification and a DNA-templated reaction to achieve a dual readout with either fluorescence (microtiter plate) or naked eye (lateral flow assay: LFA) detection. The nucleic acid templated reaction is based on an SN Ar that simultaneously transfers biotin from one Peptide Nucleic Acid (PNA) strand to another PNA strand, enabling LFA detection while uncaging a coumarin for fluorescence readout. This methodology has been applied to the detection of a DNA or RNA sequence uniquely attributed to the SARS-CoV-2.

Withaferin A, a polyfunctional pharmacophore that includes covalent engagement of IPO5, is an inhibitor of influenza A replication.

Withaferin A, a natural steroidal lactone found in the extracts of Withania somnifera, is used extensively in traditional medicine and part of an ancient remedy in ayurvedic medicine. Prior investigations into its mode of action have shown withaferin to be a polyfunctional pharmacophore with the covalent engagement of a multitude of therapeutic targets. Herein, we report that withaferin A is also a covalent inhibitor of IPO5, an importin that translocates cargos from the cytosol to the nucleus. We show that withaferin inhibits influenza A replication in epithelial cells (A549). Using a panel of inhibitors that selectively recapitulate part of withaferin A's pharmacological profile (goyazensolide, withaferin A derivatives, FiVe1, and bardoxolone methyl), we show that IPO5 inhibition contributes to the influenza replication inhibition but is not essential for the observed activity of withaferin A. We show that bardoxolone methyl, a semisynthetic triterpenoid in clinical development to treat chronic kidney disease and that shares some of the pharmacological profile of withaferin, also inhibits influenza A replication effectively. The inhibitory activity against influenza A replication should stimulate further studies to repurpose this therapeutic.

Suprastapled Peptides: Hybridization-Enhanced Peptide Ligation and Enforced α-Helical Conformation for Affinity Selection of Combinatorial Libraries.

Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein-protein interactions (PPIs). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA-peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide's conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53/MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads, and the best binders were identified by mass spectrometry.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

Dual Bcl-XL /Bcl-2 inhibitors discovered from DNA-encoded libraries using a fragment pairing strategy.

A dual Bcl-XL / Bcl-2 inhibitor was discovered from DNA-encoded libraries using a two steps process. In the first step, DNA was used to pair PNA-encoded fragments exploring > 250 000 combinations. In the second step, a focused library combining the selected fragments with linkers of different lengths and geometries led to the identification of tight binding adducts that were further investigated for their selective target engagement in pull-down assays, for their affinity by SPR, and their selectivity in a cytotoxicity assay. The best compound showed comparable cellular activity to venetoclax, the first-in-class therapeutic targeting Bcl-2.

Experimental Identification of Immuno- dominant B-cell Epitopes from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current public health crisis with devastating consequences to our societies. This COVID-19 pandemic has become the most serious threat to global public health in recent history. Given the unprecedented economic and social impact that it is causing, identification of immunodominant epitopes from SARS-CoV-2 is of great interest, not only to gain better insight into the adaptive immune response, but also for the development of vaccines, treatments and diagnostic tools. In this review, we summarize the already published or preprinted reports on the experimental identification of B-cell linear epitopes of SARS-CoV-2 proteins. Six different epitopes leading to neutralizing antibodies have been identified. Moreover, a summary of peptide candidates to be used for diagnostic tools is also included.

Probing for Thiol-Mediated Uptake into Bacteria.

Cellular uptake mediated by cyclic oligochalcogenides (COCs) is emerging as a conceptually innovative method to penetrate mammalian cells. Their mode of action is based on dynamic covalent oligochalcogenide exchange with cellular thiols. To test thiol-mediated uptake in bacteria, five antibiotics have been equipped with up to three different COCs: One diselenolane and two dithiolanes. We found that the COCs do not activate antibiotics in Gram-negative bacteria. In Gram-positive bacteria, the COCs inactivate antibiotics that act in the cytoplasm and reduce the activity of antibiotics that act on the cell surface. These results indicate that thiol-mediated uptake operates in neither of the membranes of bacteria. COCs are likely to exchange with thiols on the inner, maybe also on the outer membrane, but do not move on. Concerning mammalian cells, the absence of a COC-mediated uptake into bacteria observed in this study disfavors trivial mechanisms, such as passive diffusion, and supports the existence of more sophisticated, so far poorly understood uptake pathways.

PNA-Based Dynamic Combinatorial Libraries (PDCL) and screening of lectins.

Selections from dynamic combinatorial libraries (DCL) benefit from the dynamic nature of the library that can change constitution upon addition of a selection pressure, such as ligands binding to a protein. This technology has been predominantly used with small molecules interacting with each other through reversible covalent interaction. However, application of this technology in biomedical research and drug discovery has been limited by the reversibility of covalent exchange and the analytical deconvolution of small molecule fragments. Here we report a supramolecular approach based on the use of a constant short PNA tag to direct the combinatorial pairing of fragment. This PNA tag yields fast exchange kinetics, while still delivering the benefits of cooperativity, and provides favourable properties for analytical deconvolution by MALDI. A selection from >6,000 assemblies of glycans (mono-, di-, tri-saccharides) targeting AFL, a lectin from pathogenic fungus, yielded a 95 nM assembly, nearly three orders of magnitude better in affinity than the corresponding glycan alone (41 µM).

Kilian Muñiz (1970-2020).

Kilian Muñiz passed away unexpectedly on March 16th, 2020, at the age of only 49. Kilian was a leading figure in the field of catalytic (di-)amination reactions. He will be remembered as one of the finest, most passionate chemists, a dear colleague, and, most of all, as a close friend.

PNA as a Biosupramolecular Tag for Programmable Assemblies and Reactions.

The programmability of oligonucleotide hybridization offers an attractive platform for the design of assemblies with emergent properties or functions. Developments in DNA nanotechnologies have transformed our thinking about the applications of nucleic acids. Progress from designed assemblies to functional outputs will continue to benefit from functionalities added to the nucleic acids that can participate in reactions or interactions beyond hybridization. In that respect, peptide nucleic acids (PNAs) are interesting because they combine the hybridization properties of DNA with the modularity of peptides. In fact, PNAs form more stable duplexes with DNA or RNA than the corresponding natural homoduplexes. The high stability achieved with shorter oligomers (an 8-mer is sufficient for a stable duplex at room temperature) typically results in very high sequence fidelity in the hybridization with negligible impact of the ionic strength of the buffer due to the lack of electrostatic repulsion between the duplex strands. The simple peptidic backbone of PNA has been shown to be tolerant of modifications with substitutions that further enhance the duplex stability while providing opportunities for functionalization. Moreover, the metabolic stability of PNAs facilitates their integration into systems that interface with biology. Over the past decade, there has been a growing interest in using PNAs as biosupramolecular tags to program assemblies and reactions. A series of robust templated reactions have been developed with functionalized PNA. These reactions can be used to translate DNA templates into functional polymers of unprecedented complexity, fluorescent outputs, or bioactive small molecules. Furthermore, cellular nucleic acids (mRNA or miRNA) have been harnessed to promote assemblies and reactions in live cells. The tolerance of PNA synthesis also lends itself to the encoding of small molecules that can be further assembled on the basis of their nucleic acid sequences. It is now well-established that hybridization-based assemblies displaying two or more ligands can interact synergistically with a target biomolecule. These assemblies have now been shown to be functional in vivo. Similarly, PNA-tagged macromolecules have been used to prepare bioactive assemblies and three-dimensional nanostructures. Several technologies based on DNA-templated synthesis of sequence-defined polymers or DNA-templated display of ligands have been shown to be compatible with reiterative cycles of selection/amplification starting with large libraries of DNA templates, bringing the power of in vitro evolution to synthetic molecules and offering the possibility of exploring uncharted molecular diversity space with unprecedented scope and speed.

Novel PTP1B inhibitors identified by DNA display of fragment pairs.

DNA display of PNA-encoded libraries was used to pair fragments containing different phosphotyrosine surrogates with diverse triazoles. Microarray-based screening of the combinatorially paired fragment sets (62,500 combinations) against a prototypical phosphatase, PTP1B, was used to identify the fittest fragments. A focused library (10,000 members) covalently pairing identified fragments with linkers of different length and geometry was synthesized. Screening of the focused library against PTP1B and closely related TCPTP revealed orthogonal inhibitors. The selectivity of the identified inhibitors for PTP1B versus TCPT was confirmed by enzymatic inhibition assay.

Identification of Covalent Bromodomain Binders through DNA Display of Small Molecules.

The regulation of transcriptional programs by epigenetic readers (bromodomains) has been linked to the development of several pathologies. Notably, it has been implicated in the regulation of cellular growth and evasion of apoptosis, in cancer as well as in inflammation. The discovery of small-molecule probes to dissect the role of bromodomains is thus important. We demonstrate that specific cysteine residues conserved across the bromodomains can be harnessed for covalent trapping. We report the discovery of two small molecules that form a covalent bond with cysteine residues conserved across the bromodomain family, analyze the subset of bromodomains that can be addressed through covalent binding, and show proteomic analyses enabled by the enrichment of bromodomains from native lysates.

PNA-encoded synthesis (PES) of a 10 000-member hetero-glycoconjugate library and microarray analysis of diverse lectins.

Identification of selective and synthetically tractable ligands to glycan-binding proteins is important in glycoscience. Carbohydrate arrays have had a tremendous impact on profiling glycan-binding proteins and as analytical tools. We report a highly miniaturized synthetic format to access nucleic-acid-encoded hetero-glycoconjugate libraries with an unprecedented diversity in the combinations of glycans, linkers, and capping groups. Novel information about plant and bacterial lectin specificity was obtained by microarray profiling, and we show that a ligand identified on the array can be converted to a high-affinity soluble ligand by straightforward chemistry.

Synthesis of sesquiterpene-inspired derivatives designed for covalent binding and their inhibition of the NF-κB pathway.

A significant portion of bioactive secondary metabolites are endowed with reactive functionalities that can engage in covalent interactions with their target. Sesquiterpene lactones in particular are rich in Michael acceptors that react with cysteines. Several polycyclic scaffolds derived from total synthesis or readily available polycyclic terpenes were used as the starting point in the synthesis of a library aiming to project mildly reactive functionalities (Michael acceptors or chloroacetates) with diverse geometries. Screening of the library for inhibition of the NF-κB pathway revealed several potent inhibitors that are chemically readily accessible.

A LecA ligand identified from a galactoside-conjugate array inhibits host cell invasion by Pseudomonas aeruginosa.

Lectin LecA is a virulence factor of Pseudomonas aeruginosa involved in lung injury, mortality, and cellular invasion. Ligands competing with human glycoconjugates for LecA binding are thus promising candidates to counteract P. aeruginosa infections. We have identified a novel divalent ligand from a focused galactoside(Gal)-conjugate array which binds to LecA with very high affinity (Kd = 82 nM). Crystal structures of LecA complexed with the ligand together with modeling studies confirmed its ability to chelate two binding sites of LecA. The ligand lowers cellular invasiveness of P. aeruginosa up to 90 % when applied in the range of 0.05-5 µM. Hence, this ligand might lead to the development of drugs against P. aeruginosa infection.

Polyubiquitination of transforming growth factor ß (TGFß)-associated kinase 1 mediates nuclear factor-κB activation in response to different inflammatory stimuli.

The transcription factor nuclear factor κB (NF-κB) plays a central role in regulating inflammation in response to several external signals. The TGFß-associated kinase 1 (TAK1) is an upstream regulator of NF-κB signaling. In TGFß-stimulated cells, TAK1 undergoes Lys-63-linked polyubiquitination at Lys-34 by TNF receptor-associated factor 6 and is thereby activated. The aim of this study was to investigate whether TAK1 polyubiquitination at Lys-34 is also essential for NF-κB activation via TNF receptor, IL-1 receptor and toll-like receptor 4. We observed that TAK1 polyubiquitination occurred at Lys-34 and required the E3 ubiquitin ligase TNF receptor-associated factor 6 after stimulation of cells with IL-1ß. Polyubiquitination of TAK1 also occurred at Lys-34 in cells stimulated by TNF-α and LPS, which activates TLR4, as well as in HepG2 and prostate cancer cells stimulated with TGFß, which in all cases resulted in NF-κB activation. Expression of a K34R-mutant TAK1 led to a reduced NF-κB activation, IL-6 promoter activity, and proinflammatory cytokine secretion by TNF-α-stimulated PC-3U cells. Similar results were obtained in the mouse macrophage cell line RAW264.7 after LPS treatment. In conclusion, polyubiquitination of TAK1 is correlated with activation of TAK1 and is essential for activation of NF-κB signaling downstream of several receptors.

Self-assembled antibody multimers through peptide nucleic acid conjugation.

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.

Picture perfect: DNA-templated photoaffinity labeling.

Just so far apart and no further: Small molecules target ID by DNA tagging. Nucleic acid hybridization has been used to pair a photo-crosslinking group with a small molecule of interest. Following photoactivation, the protein(s) interacting with the small molecule are covalently linked to a nucleic acid tag.

Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2 Detection).

Pseudo-complementary oligonucleotides contain artificial nucleobases designed to reduce duplex formation in the pseudo-complementary pair without compromising duplex formation to targeted (complementary) oligomers. The development of a pseudo-complementary A:T base pair, Us:D, was important in achieving dsDNA invasion. Herein, we report pseudo-complementary analogues of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analogue of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), which is also cationic. We show that while complementary peptide nucleic acids (PNA) form a much more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. We show that this enables dsDNA invasion at physiological salt concentration and that stable invasion complexes are obtained with low equivalents of PNAs (2-4 equiv). We harnessed the high yield of dsDNA invasion for the detection of RT-RPA amplicon using a lateral flow assay (LFA) and showed that two strains of SARS-CoV-2 can be discriminated owing to single nucleotide resolution.

Asymmetric synthesis of pochonin†E and F, revision of their proposed structure, and their conversion to potent Hsp90 inhibitors.

A concise and modular synthesis of pochonin E and F, and their epimers at C-6 established the correct stereochemistry of these two natural products. Several members of the pochonin family have been shown to bind the heat shock protein 90 (Hsp90), which has been the focus of intense drug discovery efforts. Pochonin E and F as well as their epimers were derivatized into the corresponding pochoximes and further modified at the C-6 position. Molecular dynamics simulations, docking studies, and Hsp90 affinity measurements were performed to evaluate the impact of these modifications.