A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

From privileged natural product scaffolds to PNA-encoded chemical libraries

En croisant notre connaissance en synthèse de collections encodées par APN, notre expérience obtenue lors de la synthèse des quelques membres de la famille des lactones d'acide résorcylique, et notre constant intérêt dans l'inhibition de kinases et chaperonnes moléculaires, nous avons construit plusieurs collections moléculaires encodées par APN avec des pharmacophores variés incluant des hétérocycles, des acides aminés, des dérivés synthétiques de lactones d'acide résorcylique, et des médicaments autorisés par la FDA ou des fragments de ceux-ci, comme autant de points de diversité, dirigés contre différentes protéines cibles. La chaperonne moléculaire HSP70, les protéines contenant un bromodomaine et d'autres kinases ont toutes été exposées à ces collections encodées par APN et résolues spatialement sur micropuce ADN, démontrant ainsi la robustesse et la puissance de cette méthodologie de criblage dans une tentative de dépasser l'effet de goulot en découverte médicamenteuse

PNA-encoded chemical libraries

Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70

Facile access to modified and functionalized PNAs through Ugi-based solid phase oligomerization

Peptide nucleic acids (PNAs) derivatized with functional molecules are increasingly used in diverse biosupramolecular applications. PNAs have proven to be highly tolerant to modifications and different applications benefit from the use of modified PNAs, in particular modifications at the γ position. Herein we report simple protocols to access modified PNAs from iterative Ugi couplings which allow modular modifications at the α, β or γ position of the PNA backbone from simple starting materials. We demonstrate the utility of the method with the synthesis of several bioactive small molecules (a peptide ligand, a kinase inhibitor and a glycan)-PNA conjugates

Screening for covalent inhibitors using DNA-display of small molecule libraries functionalized with cysteine reactive moieties

DNA-encoded chemical libraries are increasingly used to identify leads for drug discovery or chemical biology. Despite the resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries containing Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The methodology was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library revealed novel covalent inhibitors for MEK2 and ERBB2

Expanding the Scope of PNA-Encoded Synthesis (PES): Mtt-Protected PNA Fully Orthogonal to Fmoc Chemistry and a Broad Array of Robust Diversity-Generating Reactions

Nucleic acid-encoded libraries are emerging as an attractive and highly miniaturized format for the rapid identification of protein ligands. An important criterion in the synthesis of nucleic acid encoded libraries is the scope of reactions that can be used to introduce molecular diversity and devise divergent pathways for diversity-oriented synthesis (DOS). To date, the protecting group strategies that have been used in peptide nucleic acid (PNA) encoded synthesis (PES) have limited the choice of reactions used in the library synthesis to just a few prototypes. Herein, we describe the preparation of PNA monomers with a protecting group combination (Mtt/Boc) that is orthogonal to Fmoc-based synthesis and compatible with a large palette of reactions that have been productively used in DOS (palladium cross-couplings, metathesis, reductive amination, amidation, heterocycle formation, nucleophilic addition, conjugate additions, Pictet–Spengler cyclization). We incorporate γ-modifications in the PNA backbone that are known to enhance hybridization and solubility. We demonstrate the robustness of this strategy with a library synthesis that is characterized by MALDI MS analysis at every step

DNA display of fragment pairs as a tool for the discovery of novel biologically active small molecules

Fragment-based lead discovery has proven to be a powerful method in the drug discovery process. The combinatorial output that is accessible by combining fragments is very attractive; however, identifying fragment pairs that bind synergistically and linking them productively can be challenging. Several technologies have now been established to prepare and screen nucleic acid-encoded libraries (ssDNA, dsDNA, PNA), and it has been shown that pairs of molecules combined by hybridization can bind synergistically to a target. Herein we apply this concept to combinatorially pair two libraries of small molecule fragments, use the fittest fragments supplemented with closely related analogs to build a focused library covalently linking the fragments with different spacers, and apply this strategy to the discovery of a potent ligand for Hsp70

Selective affinity-based probe for oncogenic kinases suitable for live cell imaging

Cell permeable probes to image the presence and localization of kinases are important in studying their function and as diagnostic tools. Despite the central role of kinases as therapeutic targets, there are remarkably few probes available. Herein we report the discovery of a probe to image two therapeutically relevant kinases: EGFR and ERBB2. The probe was identified from a library based on a scaffold derived from the resorcyclic acid lactones that form a covalent adduct by reacting specifically with an unconserved cysteine in the nucleotide-binding site of the kinases. We demonstrated the utility of the newly discovered probe by imaging of EGFR localization and ERBB2 inhibition in live cells

Recombinant Macrocyclic Lanthipeptides Incorporating Non-Canonical Amino Acids

Nisin is a complex lanthipeptide that has broad spectrum antibacterial activity. In efforts to broaden the structural diversity of this ribosomally synthesized lantibiotic, we now report the recombinant expression of Nisin variants that incorporate noncanonical amino acids (ncAAs) at discrete positions. This is achieved by expressing the <i>nisA</i> structural gene, cyclase (<i>nisC</i>) and dehydratase (<i>nisB</i>), together with an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase pair in Escherichia coli. A number of ncAAs with novel chemical reactivity were genetically incorporated into NisA, including an α-chloroacetamide-containing ncAA that allowed for the expression of Nisin variants with novel macrocyclic topologies. This methodology should allow for the exploration of lanthipeptide variants with new or enhanced activities

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