Covalent‐Allosteric Kinase Inhibitors

Targeting and stabilizing distinct kinase conformations is an instrumental strategy for dissecting conformation‐dependent signaling of protein kinases. Herein the structure‐based design, synthesis, and evaluation of pleckstrin homology (PH) domain‐dependent covalent‐allosteric inhibitors (CAIs) of the kinase Akt is reported. These inhibitors bind covalently to a distinct cysteine of the kinase and thereby stabilize the inactive kinase conformation. These modulators exhibit high potency and selectivity, and represent an innovative approach for chemical biology and medicinal chemistry research.     

High‐Throughput Kinetic Analysis for Target‐Directed Covalent Ligand Discovery

Cysteine‐reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high‐quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan‐reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine‐reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2‐selective allosteric (type IV) kinase inhibitor whose novel mode‐of‐action could be exploited therapeutically.     

Fuel‐Responsive Allosteric DNA‐Based Aptamers for the Transient Release of ATP and Cocaine

We show herein that allostery offers a key strategy for the design of out‐of‐equilibrium systems by engineering allosteric DNA‐based nanodevices for the transient loading and release of small organic molecules. To demonstrate the generality of our approach, we used two model DNA‐based aptamers that bind ATP and cocaine through a target‐induced conformational change. We re‐engineered these aptamers so that their affinity towards their specific target is controlled by a DNA sequence acting as an allosteric inhibitor. The use of an enzyme that specifically cleaves the inhibitor only when it is bound to the aptamer generates a transient allosteric control that leads to the release of ATP or cocaine from the aptamers. Our approach confirms that the programmability and predictability of nucleic acids make synthetic DNA/RNA the perfect candidate material to re‐engineer synthetic receptors that can undergo chemical fuel‐triggered release of small‐molecule cargoes and to rationally design non‐equilibrium systems.     

Mutant‐Selective Allosteric EGFR Degraders are Effective Against a Broad Range of Drug‐Resistant Mutations

Targeting epidermal growth factor receptor (EGFR) through an allosteric mechanism provides a potential therapeutic strategy to overcome drug‐resistant EGFR mutations that emerge within the ATP binding site. Here, we develop an allosteric EGFR degrader, DDC‐01‐163, which can selectively inhibit the proliferation of L858R/T790M (L/T) mutant Ba/F3 cells while leaving wildtype EGFR Ba/F3 cells unaffected. DDC‐01‐163 is also effective against osimertinib‐resistant cells with L/T/C797S and L/T/L718Q EGFR mutations. When combined with an ATP‐site EGFR inhibitor, osimertinib, the anti‐proliferative activity of DDC‐01‐163 against L858R/T790M EGFR‐Ba/F3 cells is enhanced. Collectively, DDC‐01‐163 is a promising allosteric EGFR degrader with selective activity against various clinically relevant EGFR mutants as a single agent and when combined with an ATP‐site inhibitor. Our data suggests that targeted protein degradation is a promising drug development approach for mutant EGFR.     

A Covalent Reporter of β‐Lactamase Activity for Fluorescent Imaging and Rapid Screening of Antibiotic‐Resistant Bacteria

Bacterial resistance to antibiotics poses a great clinical challenge in fighting serious infectious diseases due to complicated resistant mechanisms and time‐consuming testing methods. Chemical reaction‐directed covalent labeling of resistance‐associated bacterial proteins in the context of a complicated environment offers great opportunity for the in‐depth understanding of the biological basis conferring drug resistance, and for the development of effective diagnostic approaches. In the present study, three fluorogenic reagents LRBL1–3 for resistant bacteria labeling have been designed and prepared on the basis of fluorescence resonance energy transfer (FRET). The hydrolyzed probes could act as reactive electrophiles to attach the enzyme, β‐lactamase, and thus facilitated the covalent labeling of drug resistant bacterial strains. SDS electrophoresis and MALDI‐TOF mass spectrometry characterization confirmed that these probes were sensitive and specific to β‐lactamase and could therefore serve for covalent and localized fluorescence labeling of the enzyme structure. Moreover, this β‐lactamase‐induced covalent labeling provides quantitative analysis of the resistant bacterial population (down to 5 %) by high resolution flow cytometry, and allows single‐cell detection and direct observation of bacterial enzyme activity in resistant pathogenic species. This approach offers great promise for clinical investigations and microbiological research.     

Supramolecular Reassembly of Self‐Exfoliated Ionic Covalent Organic Nanosheets for Label‐Free Detection of Double‐Stranded DNA

Ionic covalent organic nanosheets (iCONs), a member of the two‐dimensional (2D) nanomaterials family, offer a unique functional platform for a wide range of applications. Herein, we explore the potential of an ethidium bromide (EB)‐based covalent organic framework ( EB‐TFP ) that self‐exfoliates in water resulting in 2D ionic covalent organic nanosheets ( EB‐TFP‐iCONs ) for the selective detection of double‐stranded DNA (dsDNA). In an aqueous medium, the self‐exfoliated EB‐TFP‐iCONs reassemble in the presence of dsDNA resulting in hybrid EB‐TFP‐iCONs‐DNA crystalline nanosheets with enhanced fluorescence at 600 nm. Detailed steady‐state and time‐resolved emission studies revealed that the reassembly phenomenon was highly selective for dsDNA when compared to single‐stranded DNA (ssDNA), which allowed us to use the EB‐TFP‐iCONs as a 2D fluorescent platform for the label‐free detection of complementary DNA strands.     

Arylfluorosulfate‐Based Electrophiles for Covalent Protein Labeling: A New Addition to the Arsenal

Selective covalent modification of a targeted protein is a powerful tool in chemical biology and drug discovery, with applications ranging from identification and characterization of proteins and their functions to the development of targeted covalent inhibitors. Most covalent ligands contain an affinity motif and an electrophilic warhead that reacts with a nucleophilic residue of the targeted protein. Because the electrophilic warhead is prone to react and modify off‐target nucleophiles, its reactivity should be balanced carefully to maximize target selectivity. Arylfluorosulfates have recently emerged as latent electrophiles for selective labeling of context‐specific tyrosine and lysine residues in protein pockets. Here, we review the recent but intense introduction of arylfluorosulfates into the arsenal of available warheads for selective covalent modification of proteins. We highlight the untapped potential of this functional group for use in chemical biology and drug discovery.     

Allosteric Tuning of Caspase‐7: A Fragment‐Based Drug Discovery Approach

The caspase family of cysteine proteases are highly sought‐after drug targets owing to their essential roles in apoptosis, proliferation, and inflammation pathways. High‐throughput screening efforts to discover inhibitors have gained little traction. Fragment‐based screening has emerged as a powerful approach for the discovery of innovative drug leads. This method has become a central facet of drug discovery campaigns in the pharmaceutical industry and academia. A fragment‐based drug discovery campaign against human caspase‐7 resulted in the discovery of a novel series of allosteric inhibitors. An X‐ray crystal structure of caspase‐7 bound to a fragment hit and a thorough kinetic characterization of a zymogenic form of the enzyme were used to investigate the allosteric mechanism of inhibition. This work further advances our understanding of the mechanisms of allosteric control of this class of pharmaceutically relevant enzymes, and provides a new path forward for drug discovery efforts.     

Target Enzyme‐Activated Two‐Photon Fluorescent Probes: A Case Study of CYP3A4 Using a Two‐Dimensional Design Strategy

The rapid development of fluorescent probes for monitoring target enzymes is still a great challenge owing to the lack of efficient ways to optimize a specific fluorophore. Herein, a practical two‐dimensional strategy was designed for the development of an isoform‐specific probe for CYP3A4, a key cytochrome P450 isoform responsible for the oxidation of most clinical drugs. In first dimension of the design strategy, a potential two‐photon fluorescent substrate ( NN ) for CYP3A4 was effectively selected using ensemble‐based virtual screening. In the second dimension, various substituent groups were introduced into NN to optimize the isoform‐selectivity and reactivity. Finally, with ideal selectivity and sensitivity, NEN was successfully applied to the real‐time detection of CYP3A4 in living cells and zebrafish. These findings suggested that our strategy is practical for developing an isoform‐specific probe for a target enzyme.     

Covalent Photosensitizer–Polyoxometalate‐Catalyst Dyads for Visible‐Light‐Driven Hydrogen Evolution

A general concept for the covalent linkage of coordination compounds to bipyridine‐functionalized polyoxometalates is presented. The new route is used to link an iridium photosensitizer to an Anderson‐type hydrogen‐evolution catalyst. This covalent dyad catalyzes the visible‐light‐driven hydrogen evolution reaction (HER) and shows superior HER activity compared with the non‐covalent reference. Hydrogen evolution is observed over periods >1 week. Spectroscopic, photophysical, and electrochemical analyses give initial insight into the stability, electronic structure, and reactivity of the dyad. The results demonstrate that the proposed linkage concept allows synergistic covalent interactions between functional coordination compounds and reactive molecular metal oxides.     

Extending the Lifetime of Native GTP‐Bound Ras for Site‐Resolved NMR Measurements: Quantifying the Allosteric Dynamics

Characterization of native GTP‐bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors, which however has been depressed by its intrinsic instability. Herein, an effective approach for extending the lifetime of Ras?GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras. This approach, combined with a postprocessing method that suppresses residual Ras?GDP signals, is applied to the site‐resolved NMR measurement of the allosteric dynamics of Ras?GTP. The observed network of concerted motions well covers the recently identified allosteric inhibitor‐binding pockets, but the motions are more confined than those of Ras?GppNHp, advocating the use of native GTP for development of allosteric inhibitors. The Sos‐based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.     

A Potent,Selective and Cell‐Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell‐active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure‐based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC₅₀=31±2 nM , K_D=53±2 nM ) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non‐epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3‐SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well‐characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease.     

Hydrogen‐Bond‐Driven Controlled Molecular Marriage in Covalent Cages

A supramolecular approach that uses hydrogen‐bonding interaction as a driving force to accomplish exceptional self‐sorting in the formation of imine‐based covalent organic cages is discussed. Utilizing the dynamic covalent chemistry approach from three geometrically similar dialdehydes ( A , B , and D ) and the flexible triamine tris(2‐aminoethyl)amine ( X ), three new [3+2] self‐assembled nanoscopic organic cages have been synthesized and fully characterized by various techniques. When a complex mixture of the dialdehydes and triamine X was subjected to reaction, it was found that only dialdehyde B (which has OH groups for H‐bonding) reacted to form the corresponding cage B₃X₂ selectively. Surprisingly, the same reaction in the absence of aldehyde B yielded a mixture of products. Theoretical and experimental investigations are in complete agreement that the presence of the hydroxyl moiety adjacent to the aldehyde functionality in B is responsible for the selective formation of cage B₃X₂ from a complex reaction mixture. This spectacular selection was further analyzed by transforming a nonpreferred (non‐hydroxy) cage into a preferred (hydroxy) cage B₃X₂ by treating the former with aldehyde B . The role of the H‐bond in partner selection in a mixture of two dialdehydes and two amines has also been established. Moreover, an example of unconventional imine bond metathesis in organic cage‐to‐cage transformation is reported.     

A Potent,Selective and Cell‐Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3)

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is essential for maturation of ribosomes, may have a role in lipogenesis, and is implicated in several diseases. A potent, selective, and cell‐active PRMT3 inhibitor would be a valuable tool for further investigating PRMT3 biology. Here we report the discovery of the first PRMT3 chemical probe, SGC707, by structure‐based optimization of the allosteric PRMT3 inhibitors we reported previously, and thorough characterization of this probe in biochemical, biophysical, and cellular assays. SGC707 is a potent PRMT3 inhibitor (IC₅₀=31±2 nM , K_D=53±2 nM ) with outstanding selectivity (selective against 31 other methyltransferases and more than 250 non‐epigenetic targets). The mechanism of action studies and crystal structure of the PRMT3‐SGC707 complex confirm the allosteric inhibition mode. Importantly, SGC707 engages PRMT3 and potently inhibits its methyltransferase activity in cells. It is also bioavailable and suitable for animal studies. This well‐characterized chemical probe is an excellent tool to further study the role of PRMT3 in health and disease.     

Synthesis of a Bis‐Urea Dimer and Its Effects on the Physical Properties of an Amphiphilic Tris‐Urea Supramolecular Hydrogel

The successful development of stiff supramolecular gels is an important goal toward their practical application. One approach to stiffen supramolecular gels is to introduce covalent cross‐links. The bis‐urea dimer 2 , having a structure similar to that of the low‐molecular‐weight gelator 1 , was synthesized. Supramolecular hydrogels were formed from mixtures of 1 and 2 in appropriate ratios, with 2 acting as a covalent cross‐linker to connect the fibrous aggregates formed by the self‐assembly of 1 . The introduction of these covalent cross‐links greatly influenced the dynamic viscoelasticity of the supramolecular hydrogels. In the supramolecular hydrogel of 1 mixed with 5 % 2 , the storage modulus was 1.35 times higher than that of the supramolecular hydrogel of 1 alone, and the crossover strain was extended from 5 % to over 20 %. The supramolecular hydrogel of 1 and 2 was free‐standing and supported 13 times its own weight.     

Recovery of biological active catechol‐O‐methyltransferase isoforms from Q‐sepharose

The development of new catechol‐O‐methyltransferase inhibitors has led to an improvement in the treatment of Parkinson's disease. However, despite the fact that the soluble isoform has been extensively investigated, few studies have been published concerning membrane isoform chromatographic recovery and bioactivity levels. In this work, chromatographic profiles of both catechol‐O‐methyltransferase isoforms were compared using quaternary amine as a ligand to evaluate its activity levels and recovery rates. Results show that both proteins required different conditions for adsorption; the soluble isoform adsorption was performed at low ionic strength, while the membrane isoform required increasing linear salt gradient. However, the application of 0.5% Triton X‐100 promoted membrane isoform adsorption even at low ionic strength. Indeed, chromatographic conditions of both isoforms became similar when detergents were applied. The developed methods also appear to be highly effective in bioactivity recovery, presenting rates of 107% for soluble protein and 67 and 91% for membrane isoform without and with detergents, respectively. The chromatographic strategies with and without detergents resulted in a 4.3‐ and sevenfold purification, respectively, corresponding to specific activity values of 331 and 496 nmol/h/mg. Thus, the use of Q‐sepharose as anion exchanger was effective in the recovery of both enzymes, which is a requirement for further kinetic and pharmacological trials.     

Modern Aspects of Pseudohalogen Chemistry: News from CN‐ and PN‐Chemistry

The pseudohalogen concept still provides a powerful tool to understand the correlation between chemical properties, structure and bonding of pseudohalogen species. Starting from a modern version of the pseudohalogen concept, this research report gives an overview on different subjects of CN‐ and PN‐chemistry dealt with in our group. One of our fundamental interests lies in the synthesis and study of structure and bonding of resonance stabilized pseudohalides such as methanides, amides or binary PN‐pseudohalides. The first chapter describes new syntheses of alkaline dinitroso‐ and nitro(nitroso)cyanomethanides, methanide‐based ionic liquids and their chemical properties. The second chapter deals with azaphospholes which can be considered (i) as resonance stabilized dimers of P‐analogues of covalent azides in case of the triazadiphospholes or (ii) as [3+2] cycloaddition product of a hidden PNN 1,3 dipole (P‐analogue of a covalent azide) with dipolarophiles possessing NN or PN moieties. Starting from a [3+2] synthetic tool kit, new PN‐heterocycles (triazadiphospholes and tetrazaphospholes) and a new synthetic approach called GaCl₃‐assisted [3+2] cycloaddition are discussed.     

On‐Surface Synthesis and Characterization of Triply Fused Porphyrin–Graphene Nanoribbon Hybrids

On‐surface synthesis offers a versatile approach to prepare novel carbon‐based nanostructures that cannot be obtained by conventional solution chemistry. Graphene nanoribbons (GNRs) have potential for a variety of applications. A key issue for their application in molecular electronics is in the fine‐tuning of their electronic properties through structural modifications, such as heteroatom doping or the incorporation of non‐benzenoid rings. In this context, the covalent fusion of GNRs and porphyrins (Pors) is a highly appealing strategy. Herein we present the selective on‐surface synthesis of a Por–GNR hybrid, which consists of two Pors connected by a short GNR segment. The atomically precise structure of the Por–GNR hybrid has been characterized by bond‐resolved scanning tunneling microscopy (STM) and noncontact atomic force microscopy (nc‐AFM). The electronic properties have been investigated by scanning tunneling spectroscopy (STS), in combination with DFT calculations, which reveals a low electronic gap of 0.4 eV.     

Molecular Recognition‐Mediated Transformation of Single‐Chain Polymer Nanoparticles into Crosslinked Polymer Films

We describe single‐chain polymer nanoparticles (SCNPs) possessing intramolecular dynamic covalent crosslinks that can transform into polymer films through a molecular recognition‐mediated crosslinking process. The SCNPs utilise molecular recognition with surface‐immobilised proteins to concentrate upon a substrate, bringing the SCNPs into close spatial proximity with one another and allowing their dynamic covalent crosslinkers to undergo intra‐ to interpolymer chain crosslinking leading to the formation of polymeric film. SCNPs must possess both the capacity for specific molecular recognition and a dynamic nature to their intramolecular crosslinkers to form polymer films, and an investigation of the initial phase of film formation indicates it proceeds from features which form upon the surface then grow predominantly in the xy directions. This approach to polymer film formation presents a potential method to “wrap” surfaces displaying molecular recognition motifs—which could potentially include viral, cellular and bacterial surfaces or artificial surfaces displaying multivalent recognition motifs—within a layer of polymer film.     

Design of Turn‐On Near‐Infrared Fluorescent Probes for Highly Sensitive and Selective Monitoring of Biopolymers

Simple, sensitive, and selective detection of specific biopolymers is critical in a broad range of biomedical and technological areas. We present a design of turn‐on near‐infrared (NIR) fluorescent probes with intrinsically high signal‐to‐background ratio. The fluorescent signal generation mechanism is based on the aggregation/de‐aggregation of phthalocyanine chromophores controlled by selective binding of small‐molecule “anchor” groups to a specific binding site of a target biopolymer. As a proof‐of‐concept, we demonstrate a design of a sensor for EGFR tyrosine kinase—an important target in cancer research. The universality of the fluorescent signal generation mechanism, as well as the dependence of the response selectivity on the choice of the small‐molecule “anchor” group, make it possible to use this approach to design reliable turn‐on NIR fluorescent sensors for detecting specific protein targets present in the low‐nanomolar concentration range.