Discovery of Potent Degraders of the Dengue Virus Envelope Protein.

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here, it is reported that the development of small molecule degraders of the envelope (E) protein of dengue virus. Two classes of bivalent E-degraders are developed by linking two previously reported E-binding small molecules, GNF-2, and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E-degrader with ABL inhibitory activity while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof of concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class of direct-acting antiviral drugs.

Discovery of bivalent small molecule degraders of cyclin-dependent kinase 7 (CDK7).

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

A Coordinating Small Organic Molecule with Tunable Lower Critical Solution Temperature for Efficient Management of Solar Radiation.

Structurally well-defined small molecules with lower critical solution temperature (LCST) behavior offer enormous prospects for fine-tuning their phase transition properties to be "on-demand" applied in the specific scene but are still underexplored. Herein, a novel amphiphilic small LCST molecule is rationally designed and synthesized. The molecule, namely TG, features a conjugation of multiple short ethylene glycol (EG) chains with the functional coordinating terpyridine (Tpy) moiety. The molecule TG demonstrates excellent LCST behavior down to 0.05 × 10-3 m in a water solution. And a cloud point Tcp = 30.9 °C with a very short thermal hysteresis ΔT = 0.2 °C and good reversibility can be achieved when c = 0.1 × 10-3 m. The excellent LCST properties of TG have enabled its successful performance as the smart window for solar radiation management with the ∆Tlum, ∆TIR, and ∆Tsol being 83.6%, 49.1%, and 67.2%, respectively. Moreover, the presence of Tpy moiety in TG enables its coordination with Ru3+ and the resulting complex also exhibits modulated LCST behavior with different concentration-dependent Tcp. These studies would provide novel small-molecule-based scaffolds for constructing better solar radiation management systems as well as other thermal-responsive smart materials.

Discovery of Potent Degraders of the Dengue Virus Envelope Protein.

Targeted protein degradation has been widely adopted as a new approach to eliminate both established and previously recalcitrant therapeutic targets. Here we report the development of small molecule degraders of the envelope (E) protein of dengue virus. We developed two classes of bivalent E-degraders, linking two previously reported E-binding small molecules, GNF-2 and CVM-2-12-2, to a glutarimide-based recruiter of the CRL4CRBN ligase to effect proteosome-mediated degradation of the E protein. ZXH-2-107 (based on GNF-2) is an E degrader with ABL inhibition while ZXH-8-004 (based on CVM-2-12-2) is a selective and potent E-degrader. These two compounds provide proof-of-concept that difficult-to-drug targets such as a viral envelope protein can be effectively eliminated using a bivalent degrader and provide starting points for the future development of a new class antiviral drugs.

Tunable Fluorescence and Morphology of Aggregates Built from a Mechanically Bonded Amphiphilic Bistable [2]Rotaxane.

Advanced Organic Chemical Materials Co-constructed Mechanically bonded amphiphiles (MBAs), also known as mechanically interlocked molecules (MIMs), have emerged as an important kind of functional building block for the construction of artificial molecular machines and soft materials. Herein, a novel MBA, i. e., bistable [2]rotaxane H2 was designed and synthesized. In the solution state, H2 demonstrated pH and metal ion-responsive emissions due to the presence of a distance-dependent photoinduced electron transfer (PET) process and the fluorescence resonance energy transfer (FRET) process, respectively. Importantly, the amphiphilic feature of H2 has endowed it with unique self-assembly capability, and nanospheres were obtained in a mixed H2 O/CH3 CN solvent. Moreover, the morphology of H2 aggregates can be tuned from nanospheres to vesicles due to the pH-controlled shuttling motion-induced alternation of H2 amphiphilicity. Interestingly, larger spheres with novel pearl-chain-like structures from H2 were observed after adding stoichiometric Zn2+ . In particular, H2 shows pH-responsive emissions in its aggregation state, allowing the visualization of the shuttling movement by just naked eyes. It is assumed that the well-designed [2]rotaxane, and particularly the proposed concept of MBA shown here, will further enrich the families of MIMs, offering prospects for synthesizing more MIMs with novel assembly capabilities and bottom-up building dynamic smart materials with unprecedented functions.

Zwitterionic nanocapsule-based wound dressing with the function of gradient release of multi-drugs for efficient wound healing.

Efficient wound healing has attracted great interest due to the prevalence of skin damage. It is still highly desired yet challenging to construct a multi-drug loaded wound dressing that can release different drugs at different times to meet specific requirements towards different healing stages. Herein, a wound dressing was developed based on thermoresponsive zwitterionic nanocapsules (ZNs) that were sandwiched between two double-layered fabrics to regulate the multiple drug release pathway. The salt-response of the obtained ZNs was greatly suppressed while its transition temperature was regulated to be ∼37 °C to fit the needs of the physiological environment. Two bioactive substances, human basic fibroblast growth factor (bFGF) for tissue regeneration and norfloxacin for anti-inflammation, were loaded in the ZNs and on the surface of fabrics, respectively, to achieve separative gradient release. The in vitro drug release tests revealed that norfloxacin could be released relatively fast (∼24 h) while the release rate of bFGF was much slower (∼168 h), matching the specific time requirements of inflammation and proliferation stages very well. The in vivo wound healing experiment also confirmed the high wound healing efficiency of the wound dressing developed here, compared to the wound dressings without gradient release characteristics. We believe the strategy illustrated here will provide new insights into the design and biomedical applications of zwitterionic nanocapsules.

Polysaccharide-Based Injectable Hydrogels with Fast Gelation and Self-Strengthening Mechanical Kinetics for Oral Tissue Regeneration.

Oral defects lead to a series of function disorders, severely threatening the patients' health. Although injectable hydrogels are widely studied in tissue regeneration, their mechanical performance is usually stationary after implant, without further self-adaption toward the microenvironment. Herein, an injectable hydrogel with programmed mechanical kinetics of instant gelation and gradual self-strengthening along with outstanding biodegradation ability is developed. The fast gelation is realized through rapid Schiff base reaction between biodegradable chitosan and aldehyde-modified sodium hyaluronate, while self-strengthening is achieved via slow reaction between redundant amino groups on chitosan and epoxy-modified hydroxyapatite. The resultant hydrogel also possesses multiple functions including (1) bio-adhesion, (2) self-healing, (3) bactericidal, (4) hemostasis, and (5) X-ray in situ imaging, which can be effectively used for oral jaw repair. We believe that the strategy illustrated here will provide new insights into dynamic mechanical regulation of injectable hydrogels and promote their application in tissue regeneration.

Mechanism of the Impact-Sensitivity Reduction of Energetic CL-20/TNT Cocrystals: A Nonequilibrium Molecular Dynamics Study.

High-energy low-sensitivity explosives are research objectives in the field of energetic materials, and the formation of cocrystals is an important method to improve the safety of explosives. However, the sensitivity reduction mechanism of cocrystal explosives is still unclear. In this study, CL-20/TNT, CL-20 and TNT crystals were taken as research objects. On the basis of the ReaxFF-lg reactive force field, the propagation process of the wave front in the crystals at different impact velocities was simulated. The molecular dynamics data were used to analyze the molecular structure changes and initial chemical reactions, and to explore the sensitivity reduction mechanism of the CL-20/TNT cocrystal. The results showed that the chemical reaction of the CL-20/TNT cocrystal, compared with the CL-20 single crystal, is different under different impact velocities. At an impact velocity of 2 km/s, polymerization and separation of the component molecules weakened the decomposition of CL-20. At an impact velocity of 3 km/s, the decay rates of CL-20 and TNT in the cocrystal decreased, and the intermediate products were enhanced, such as nitrogen oxides. At an impact velocity of 4 km/s, the cocrystal had little effect on the decay rates of the molecules and formation of CO2, but it enhanced formation of N2 and H2O. This may explain the reason for the impact-sensitivity reduction of the CL-20/TNT cocrystal.

Advancing targeted protein degrader discovery by measuring cereblon engagement in cells.

Measurement of target engagement in cells is critical to understand the molecular pharmacology of drugs and chemical probes. Many targeted protein degraders engage the E3 ligase CRL4CRBN and induce proximity with target neosubstrates resulting in their polyubiquitination and subsequent proteasomal degradation. Here we describe the development of a sensitive and robust cellular NanoBRET-based assay that measures occupancy of the CRBN ligand binding site. The assay is based on a bioluminescence resonance energy transfer (BRET) between NanoLuc luciferase tagged CRBN and a BODIPY-lenalidomide tracer which can be competed out by CRBN ligands, including PROTACs and molecular glues. The assay is compatible with a 384-well plate setup, does not require transfections and can be performed in a single day with only 3-4h of laboratory time. The protocols can be used to design other NanoLuc fusion engagement assays based on BODIPY tracers.

Exploring the target scope of KEAP1 E3 ligase-based PROTACs.

Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.

Design of novel temperature-resistant and salt-tolerant acrylamide-based copolymers by aqueous dispersion polymerization.

Development of polymer-based flooding technology to improve oil recovery efficiency, water dispersion copolymerization of acrylamide, cationic monomer methacryloxyethyltrimethyl ammonium chloride (METAC), and anionic monomer acrylic acid (AA) were carried out in aqueous ammonium sulfate solution with polyvinyl pyrrolidone (PVP) as the stabilizer. The copolymers were characterized by 1H-NMR, FT-IR, TG, and SEM to confirm that they were prepared successfully and exhibited excellent salt-resistant property. Moreover, the effect of the aqueous solution of ammonium sulfate (AS) concentration, stabilizer concentration, and initiator concentration on the viscosity and size were systematically investigated. To further improve the thermal endurance properties of copolymer, hydrophobic monomers with different alkyl chain lengths were added to the above system. The acrylamide-based quadripolymer possessed prominent thermal and salt endurance properties by utilizing the advantages of zwitterionic structure and hydrophobic monomer. With the temperature rising, the viscosity retention could reach 70.2% in the water and 63.8% in the saline. This work had expected to provide a new strategy to design polymers with excellent salinity tolerance and thermal-resistance performances.

Selective degradation-inducing probes for studying cereblon (CRBN) biology.

Targeted protein degradation represents a rapidly growing area in drug discovery and development. Moreover, small molecules that induce the targeted degradation of a given protein also represent an important addition to the chemical probes toolbox as these compounds can achieve selective protein knockdown, thus providing an approach that is orthogonal to genetic knockdowns. In order to develop degradation-inducing chemical probes for studying cereblon (CRBN) biology, we generated six CRBN-CRBN (homo-PROTAC) degraders and six CRBN-VHL (hetero-PROTAC) degraders. From these compounds we identified two potent and selective CRBN degraders (ZXH-4-130 and ZXH-4-137), both of which are CRBN-VHL compounds. We characterized these lead degraders by quantitative proteomics in five cell lines (MM1.S, Kelly, SK-N-DZ, HEK293T, and MOLT-4) and observed high selectivity for CRBN in all cell lines. Furthermore, we directly compared our compounds to current lead CRBN degraders and demonstrated how these probes can be used as chemical knockdown reagents for studying CRBN-dependent processes. Overall, our work provides a roadmap for thorough degrader characterization by combination western and proteomic analysis, as illustrated by the identification of ZXH-4-130 and ZXH-4-137 as CRBN-knockdown tool compounds suitable for cell-based studies.

Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2.

Aberrant activation of FGFR signaling occurs in many cancers, and ATP-competitive FGFR inhibitors have received regulatory approval. Despite demonstrating clinical efficacy, these inhibitors exhibit dose-limiting toxicity, potentially due to a lack of selectivity amongst the FGFR family and are poorly tolerated. Here, we report the discovery and characterization of DGY-09-192, a bivalent degrader that couples the pan-FGFR inhibitor BGJ398 to a CRL2VHL E3 ligase recruiting ligand, which preferentially induces FGFR1&2 degradation while largely sparing FGFR3&4. DGY-09-192 exhibited two-digit nanomolar DC50 s for both wildtype FGFR2 and several FGFR2-fusions, resulting in degradation-dependent antiproliferative activity in representative gastric cancer and cholangiocarcinoma cells. Importantly, DGY-09-192 induced degradation of a clinically relevant FGFR2 fusion protein in a xenograft model. Taken together, we demonstrate that DGY-09-192 has potential as a prototype FGFR degrader.

Selective Degradation of GSPT1 by Cereblon Modulators Identified via a Focused Combinatorial Library.

Cereblon (CRBN) is an E3 ligase adapter protein that can be reprogrammed by imide-class compounds such as thalidomide, lenalidomide, and pomalidomide to induce the degradation of neo-substrate proteins. In order to identify additional small molecule CRBN modulators, we implemented a focused combinatorial library approach where we fused an imide-based CRBN-binding pharmacophore to a heterocyclic scaffold, which could be further elaborated. We screened the library for CRBN-dependent antiproliferative activity in the multiple myeloma cell line MM1.S and identified five hit compounds. Quantitative chemical proteomics of hit compounds revealed that they induced selective degradation of GSPT1, a translation termination factor that is currently being explored as a therapeutic target for the treatment of acute myeloid leukemia. Molecular docking studies with CRBN and GSPT1 followed by analogue synthesis identified a possible hydrogen bond interaction with the central pyrimidine ring as a molecular determinant of hit compounds' selectivity. This study demonstrates that a focused combinatorial library design, phenotypic screening, and chemical proteomics can provide a suitable workflow to efficiently identify novel CRBN modulators.

Structure and Characterization of a Covalent Inhibitor of Src Kinase.

Unregulated Src activity promotes malignant processes in cancer, but no Src-directed targeted therapies are used clinically, possibly because early Src inhibitors produce off-target effects leading to toxicity. Improved selective Src inhibitors may enable Src-directed therapies. Previously, we reported an irreversible Src inhibitor, DGY-06-116, based on the hybridization of dasatinib and a promiscuous covalent kinase probe SM1-71. Here, we report biochemical and biophysical characterization of this compound. An x-ray co-crystal structure of DGY-06-116: Src shows a covalent interaction with the kinase p-loop and occupancy of the back hydrophobic kinase pocket, explaining its high potency, and selectivity. However, a reversible analog also shows similar potency. Kinetic analysis shows a slow inactivation rate compared to other clinically approved covalent kinase inhibitors, consistent with a need for p-loop movement prior to covalent bond formation. Overall, these results suggest that a strong reversible interaction is required to allow sufficient time for the covalent reaction to occur. Further optimization of the covalent linker may improve the kinetics of covalent bond formation.

A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging.

Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.

Structure-Based Design of a Potent and Selective Covalent Inhibitor for SRC Kinase That Targets a P-Loop Cysteine.

SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P-loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) SM1-71 as a starting point, we developed covalent inhibitor 15a, which discriminates SRC from other covalent targets of SM1-71 including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound 15a exhibited sustained inhibition of SRC signaling both in vitro and in vivo. Moreover, 15a exhibited potent antiproliferative effects in nonsmall cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.

Small molecule degraders of the hepatitis C virus protease reduce susceptibility to resistance mutations.

Targeted protein degradation is a promising drug development paradigm. Here we leverage this strategy to develop a new class of small molecule antivirals that induce proteasomal degradation of viral proteins. Telaprevir, a reversible-covalent inhibitor that binds to the hepatitis C virus (HCV) protease active site is conjugated to ligands that recruit the CRL4CRBN ligase complex, yielding compounds that can both inhibit and induce the degradation of the HCV NS3/4A protease. An optimized degrader, DGY-08-097, potently inhibits HCV in a cellular infection model, and we demonstrate that protein degradation contributes to its antiviral activity. Finally, we show that this new class of antiviral agents can overcome viral variants that confer resistance to traditional enzymatic inhibitors such as telaprevir. Overall, our work provides proof-of-concept that targeted protein degradation may provide a new paradigm for the development of antivirals with superior resistance profiles.

Quinoline and thiazolopyridine allosteric inhibitors of MALT1.

Quinolines and thiazolopyridines were developed as allosteric inhibitors of MALT1, with good cellular potency and exquisite selectivity. Mouse pharmacokinetic (PK) profiling showed these to have low in vivo clearance, and moderate oral exposure. The thiazolopyridines were less lipophilic than the quinolines, and one thiazolopyridine example was active in our hIL10 mouse pharmacodynamic (PD) model upon oral dosing.

Peptide-based covalent inhibitors of MALT1 paracaspase.

Potent and selective substrate-based covalent inhibitors of MALT1 protease were developed from the tetrapeptide tool compound Z-VRPR-fmk. To improve cell permeability, we replaced one arginine residue. We further optimized a series of tripeptides and identified compounds that were potent in both a GloSensor reporter assay measuring cellular MALT1 protease activity, and an OCI-Ly3 cell proliferation assay. Example compounds showed good overall selectivity towards cysteine proteases, and one compound was selected for further profiling in ABL-DLBCL cells and xenograft efficacy models.