Bioinspired Selenium-Nitrogen Exchange (SeNEx) Click Chemistry Suitable for Nanomole-Scale Medicinal Chemistry and Bioconjugation.

Click chemistry is a powerful molecular assembly strategy for rapid functional discovery. The development of click reactions with new connecting linkage is of great importance for expanding the click chemistry toolbox. We report the first selenium-nitrogen exchange (SeNEx) click reaction between benzoselenazolones and terminal alkynes (Se-N to Se-C), which is inspired by the biochemical SeNEx between Ebselen and cysteine (Cys) residue (Se-N to Se-S). The formed selenoalkyne connection is readily elaborated, thus endowing this chemistry with multidimensional molecular diversity. Besides, this reaction is modular, predictable, and high-yielding, features fast kinetics (k2≥14.43 M-1 s-1), excellent functional group compatibility, and works well at miniaturization (nanomole-scale), opening up many interesting opportunities for organo-Se synthesis and bioconjugation, as exemplified by sequential click chemistry (coupled with ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) and sulfur-fluoride exchange (SuFEx)), selenomacrocycle synthesis, nanomole-scale synthesis of Se-containing natural product library and DNA-encoded library (DEL), late-stage peptide modification and ligation, and multiple functionalization of proteins. These results indicated that SeNEx is a useful strategy for new click chemistry developments, and the established SeNEx chemistry will serve as a transformative platform in multidisciplinary fields such as synthetic chemistry, material science, chemical biology, medical chemistry, and drug discovery.

AKR7A3 modulates the metastasis of pancreatic ductal adenocarcinoma through regulating PHGDH-suppressed autophagy.

AKR7A3 is a member of the aldo-keto reductase (AKR) protein family, whose primary purpose is to reduce aldehydes and ketones to generate primary and secondary alcohols. It has been reported that AKR7A3 is downregulated in pancreatic cancer (PC). However, the mechanism underlying the effects of AKR7A3 in PC remains largely unclarified. Here, we explored the biological function, molecular mechanism and clinical relevance of AKR7A3 in pancreatic ductal adenocarcinoma (PDAC). AKR7A3 expression was downregulated in PDAC compared with adjacent normal tissues, and the lower AKR7A3 expression was related to poor prognosis. In addition, our results demonstrated that AKR7A3 could be a potential diagnostic marker for PDAC, especially in the early stages. Knockdown of AKR7A3 promoted PDAC progression and chemoresistance, while inhibiting autophagy flux. Mechanistically, AKR7A3 affected the metastasis, autophagy, and chemoresistance of PDAC by regulating PHGDH. Overall, the present study suggests that AKR7A3 inhibits PDAC progression by regulating PHGDH-induced autophagy. In addition, AKR7A3 inhibits chemoresistance via regulating PHGDH and may serve as a new therapeutic target for PDAC.

Enolate-Azide [3 + 2]-Cycloaddition Reaction Suitable for DNA-Encoded Library Synthesis.

The DNA-encoded chemical library (DEL) is a powerful hit selection technique in either basic science or innovative drug discovery. With the aim to circumvent the issue concerning DNA barcode damage in a conventional on-DNA copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), we have successfully developed the first DNA-compatible enolate-azide [3 + 2] cycloaddition reaction. The merits of this DEL chemistry include metal-free reaction and high DNA fidelity, high conversions and easy operation, broad substrate scope, and ready access to the highly substituted 1,4,5-trisubstituted triazoles. Thus, it will not only further enrich the DEL chemistry toolbox but also will have great potential in practical DEL synthesis.

NUCKS1 promotes the progression of colorectal cancer via activating PI3K/AKT/mTOR signaling pathway.

Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) is highly expressed in a variety of malignant tumors and functions as an oncogene; however, its role in colorectal cancer (CRC) remains unclear. We aimed to explore the function and regulatory mechanisms of NUCKS1 and potential therapeutic agents targeting NUCKS1 in CRC. We knocked down and overexpressed NUCKS1 in CRC cells and explored its effects in vitro and in vivo. Flow cytometry, CCK-8, Western blotting, colony formation, immunohistochemistry, in vivo tumorigenic, and transmission electron microscopy analyses were performed to determine the effects of NUCKS1 on CRC cell function. LY294002 was used to examine the mechanism of NUCKS1 expression in CRC cells. Potential therapeutic agents for NUCKS1-high CRC patients were analyzed using the CTRP and PRISM datasets, and the function of selected agents was determined by CCK-8 and Western blotting. We revealed that NUCKS1 was highly expressed in CRC tissues and clinically correlated with poor prognosis in CRC patients. NUCKS1 knockdown induces cell cycle arrest, inhibits CRC cell proliferation, and promotes apoptosis and autophagy. These results were reversed when NUCKS1 was overexpressed. Mechanistically, NUCKS1 exerts a cancer-promoting function by activating the PI3K/AKT/mTOR signaling pathway. This was reversed when LY294002 was used to inhibit the PI3K/AKT pathway. Furthermore, we determined that mitoxantrone exhibited high drug sensitivity in NUCKS1-overexpressing CRC cells. This work demonstrated NUCKS1 plays a crucial role in CRC progression via the PI3K/AKT/mTOR signaling pathway. Additionally, mitoxantrone may be a potential therapeutic agent for CRC treatment. Therefore, NUCKS1 represents a promising anti-tumor therapeutic target.

Harnessing coronavirus spike proteins' binding affinity to ACE2 receptor through a novel baculovirus surface display system.

Coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerging infectious disease currently spreading across the world. The spike (S) protein plays a key role in the receptor recognition and cell membrane fusion, making it an important target for developing vaccines, therapeutic antibodies and diagnosis. In this study, we constructed a baculovirus surface display system that efficiently presents both SARS-CoV and SARS-CoV-2 S proteins (including ectodomain, S1 subunit and receptor-binding-domain, RBD) on the surface of recombinant baculoviruses, utilizing transmembrane anchors from gp64 (signal peptide) and vesicular stomatitis virus (VSV). These recombinant baculoviruses were capable of transducing engineered HEK 293T cells overexpressing ACE2 receptors with significantly higher transduction efficiencies, indicating that S proteins displayed on baculovirus surface have antigenicity and can recognize and bind ACE2 receptors. Additionally, the transduction of SARS-CoV-2 S proteins can be inhibited by an antibody against the SARS-CoV-2 RBD. These results demonstrate that this baculovirus surface display system is a promising tool for developing antibodies, vaccines and recombinant protein production.

Discovery of potential small molecular SARS-CoV-2 entry blockers targeting the spike protein.

An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant KD) of 0.0947 µM and anti-virus IC50 of 85.75 µM.

TMEM43 promotes pancreatic cancer progression by stabilizing PRPF3 and regulating RAP2B/ERK axis.

BACKGROUND: Transmembrane protein 43 (TMEM43), a member of the transmembrane protein subfamily, plays a critical role in the initiation and development of cancers. However, little is known concerning the biological function and molecular mechanisms of TMEM43 in pancreatic cancer. METHODS: In this study, TMEM43 expression levels were analyzed in pancreatic cancer samples compared with control samples. The relationship of TMEM43 expression and disease-free survival (DFS) and overall survival (OS) were assessed in pancreatic cancer patients. In vitro and in vivo assays were performed to explore the function and role of TMEM43 in pancreatic cancer. Coimmunoprecipitation (co-IP) followed by protein mass spectrometry was applied to analyze the molecular mechanisms of TMEM43 in pancreatic cancer. RESULTS: We demonstrated that TMEM43 expression level is elevated in pancreatic cancer samples compared with control group, and is correlated with poor DFS and OS in pancreatic cancer patients. Knockdown of TMEM43 inhibited pancreatic cancer progression in vitro, decreased the percentage of S phase, and inhibited the tumorigenicity of pancreatic cancer in vivo. Moreover, we demonstrated that TMEM43 promoted pancreatic cancer progression by stabilizing PRPF3 and regulating the RAP2B/ERK axis. CONCLUSIONS: The present study suggests that TMEM43 contributes to pancreatic cancer progression through the PRPF3/RAP2B/ERK axis, and might be a novel therapeutic target for pancreatic cancer.

HSPA5 repressed ferroptosis to promote colorectal cancer development by maintaining GPX4 stability.

Colorectal cancer (CRC) is one of the most malignant cancers and its pathological mechanism is largely unknown. Unfolded protein response and ferroptosis are both critical factors involved in CRC development. However, their relationship in CRC remains to be explored. In this study, erastin was used to induce ferroptosis in CRC cells. Ferroptosis was confirmed by the detection of glutathione, malondialdehyde, and lipid reactive oxygen species. The CRC datasets were analyzed using the R software, GEPIA2, and TIMER2.0. The results indicated that GPX4 was decreased when treated with the ferroptosis inducer erastin. As an intrinsic protective pathway, the unfolded protein response was activated and HSPA5 was increased during ferroptosis. HSPA5 was found to attenuate erastin-induced GPX4 decrease, repress ferroptosis, and promote CRC cell growth both in vitro and in vivo. Mechanistically, HSPA5 bound directly to GPX4 and the interaction between HSPA5 and GPX4 increased when treated with erastin for a short time period. Although the HSPA5-GPX4 interaction failed to completely reverse erastin-induced GPX4 decrease, HSPA5 slowed down the GPX4 degradation process and gave CRC cells more time to adjust to erastin toxicity. Additionally, HSPA5 was demonstrated to play a diagnostic role and correlated to the immune microenvironment in CRC patients. Our study demonstrates that increased HSPA5 was an intrinsic protective strategy to resist ferroptosis. Specifically, HSPA5 restrained ferroptosis to promote colorectal cancer development by maintaining GPX4 stability. Our study provides potential diagnostic and therapeutic targets for patients with CRC.

Selenylation Chemistry Suitable for On-Plate Parallel and On-DNA Library Synthesis Enabling High-Throughput Medicinal Chemistry.

Click chemistry is a concept wherein modular synthesis is used for rapid functional discovery. To this end, continuous discovery of clickable chemical transformations is the pillar to support the development of this field. This report details the development of a clickable C3-H selenylation of indole that is suitable for on-plate parallel and DNA-encoded library (SeDEL) synthesis via bioinspired LUMO activation strategy. This reaction is modular, robust and highly site-selective, and it features a simple and mild reaction system (catalyzed by nonmetallic B(C6 F5 )3 at room temperature), high yields and excellent functional group compatibility. Using this method, a library of 1350 indole-selenides was parallel synthesized in an efficient and practical manner, enabling the rapid identification of 3 ai as a promising compound with nanomolar antiproliferative activity in cancer cells via in situ phenotypic screening. These results indicate the great potential of this new clickable selenylation reaction in high-throughput medicinal chemistry and chemical biology.

DNA-Encoded Libraries: Hydrazide as a Pluripotent Precursor for On-DNA Synthesis of Various Azole Derivatives.

DNA-encoded combinatorial chemical library (DEL) technology, an approach that combines the power of genetics and chemistry, has emerged as an invaluable tool in drug discovery. Skeletal diversity plays a fundamental importance in DEL applications, and relies heavily on novel DNA-compatible chemical reactions. We report herein a phylogenic chemical transformation strategy using DNA-conjugated benzoyl hydrazine as a common versatile precursor in azole chemical expansion of DELs. DNA-compatible reactions deriving from the common benzoyl hydrazine precursor showed excellent functional group tolerance with exceptional efficiency in the synthesis of various azoles, including oxadiazoles, thiadiazoles, and triazoles, under mild reaction conditions. The phylogenic chemical transformation strategy provides DELs a facile way to expand into various unique chemical spaces with privileged scaffolds and pharmacophores.

Magnetic graphene quantum dots facilitate closed-tube one-step detection of SARS-CoV-2 with ultra-low field NMR relaxometry.

The rapid and sensitive diagnosis of the highly contagious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is one of the crucial issues at the outbreak of the ongoing global pandemic that has no valid cure. Here, we propose a SARS-CoV-2 antibody conjugated magnetic graphene quantum dots (GQDs)-based magnetic relaxation switch (MRSw) that specifically recognizes the SARS-CoV-2. The probe of MRSw can be directly mixed with the test sample in a fully sealed vial without sample pretreatment, which largely reduces the testers' risk of infection during the operation. The closed-tube one-step strategy to detect SARS-CoV-2 is developed with home-made ultra-low field nuclear magnetic resonance (ULF NMR) relaxometry working at 118 µT. The magnetic GQDs-based probe shows ultra-high sensitivity in the detection of SARS-CoV-2 due to its high magnetic relaxivity, and the limit of detection is optimized to 248 Particles mL‒1. Meanwhile, the detection time in ULF NMR system is only 2 min, which can significantly improve the efficiency of detection. In short, the magnetic GQDs-based MRSw coupled with ULF NMR can realize a rapid, safe, and sensitive detection of SARS-CoV-2.

Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death.

Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

A DNA-encoded library for the identification of natural product binders that modulate poly (ADP-ribose) polymerase 1, a validated anti-cancer target.

Natural products have been an invaluable source of drug discovery, but their targets remain largely unknown. Natural products enriched DNA-encoded chemical libraries (nDELs) empower the researchers to rapidly and economically screen numerous natural products against various protein targets, and therefore promote the elucidation of the molecular mechanisms. In this work, we used poly (ADP-ribose) polymerase 1 (PARP1), as an example to explore the usage of nDEL for the functional natural products selection. We used late-stage modification approach to label three positive binders with unique DNA barcodes, whose dissociation constants range from sub-micromolar to micromolar. The selection criterion was set up according to the enrichment of these controls. Five natural products selected by this criterion directly bind to PARP1 in SPR, among which luteolin exhibits the highest inhibitory activity against PARP1. Moreover, luteolin selectively induces accumulation of DNA double-strand breaks and G2/M phase arrest in BRCA-deficient cells. All the findings from these investigations on luteolin support that PARP1 inhibition is one of the mechanisms for its anti-cancer activity.

A Chemistry for Incorporation of Selenium into DNA-Encoded Libraries.

Conventional direct C-H selenylation suffers from simple selenation with limited atom economy and complicated reaction system. In this work, we designed benzoselenazolone as a novel bifunctional selenide reagent for both off- and on-DNA C-H selenylation under rhodium(III) catalysis. We show that using benzoselenazolone allowed production of a series of selenylation products containing an adjacent aminoacyl group in a fast and efficient way, with high atom economy. The synthetic application of this method was demonstrated by taking advantage of the amide functionality as a nucleophile, directing group, and amide coupling partner. This work shows great potential in facilitating rapid construction of selenium-containing DNA-encoded chemical libraries (SeDELs), and lays the foundation for the development of selenium-containing drugs.

Synthesis of N-Acyl Sulfamates from Fluorosulfonates and Potassium Trimethylsilyloxyl Imidates.

An efficient and operationally simple method for the synthesis of N-acyl sulfamates from fluorosulfonates and potassium trimethylsilyloxyl imidates as amide precursor is reported. This approach showed broad substrate scope, mild and base-free reaction conditions, short reaction time, and high to excellent yields. Notably, we demonstrated the power of this reaction in the rapid late-stage functionalization of three complex phenol-containing bioactive molecules. Given the prevalence of phenol-containing drugs and building blocks, this method is applicable toward a diversity-oriented drug discovery.

Functionality-Independent DNA Encoding of Complex Natural Products.

DNA encoded chemical libraries (DELs) link the powers of genetics and chemical synthesis via combinatorial optimization. Through combinatorial chemistry, DELs can grow to the unprecedented size of billions to trillions. To take full advantage of the DEL approach, linking the power of genetics directly to chemical structures would offer even greater diversity in a finite chemical world. Natural products have evolved an incredible structural diversity along with their biological evolution. Herein, we used traditional Chinese medicines (TCMs) as examples in a late-stage modification toolbox approach to annotate these complex organic compounds with amplifiable DNA barcodes, which could be easily incorporated into a DEL. The method of end-products labeling also generates a cluster of isomers with a single DNA tag at different sites. These isomers provide an additional spatial diversity for multiple accessible pockets of targeted proteins. Notably, a novel PARP1 inhibitor from TCM has been identified from the natural products enriched DEL (nDEL).

The Disordered Region of the HCV Protein NS5A: Conformational Dynamics, SH3 Binding, and Phosphorylation.

Intrinsically disordered proteins (IDPs) perform their physiological role without possessing a well-defined three-dimensional structure. Still, residual structure and conformational dynamics of IDPs are crucial for the mechanisms underlying their functions. For example, regions of transient secondary structure are often involved in molecular recognition, with the structure being stabilized (or not) upon binding. Long-range interactions, on the other hand, determine the hydrodynamic radius of the IDP, and thus the distance over which the protein can catch binding partners via so-called fly-casting mechanisms. The modulation of long-range interactions also presents a convenient way of fine-tuning the protein's interaction network, by making binding sites more or less accessible. Here we studied, mainly by nuclear magnetic resonance spectroscopy, residual secondary structure and long-range interactions in nonstructural protein 5A (NS5A) from hepatitis C virus (HCV), a typical viral IDP with multiple functions during the viral life cycle. NS5A comprises an N-terminal folded domain, followed by a large (∼250-residue) disordered C-terminal part. Comparing nuclear magnetic resonance spectra of full-length NS5A with those of a protein construct composed of only the C-terminal residues 191-447 (NS5A-D2D3) allowed us to conclude that there is no significant interaction between the globular and disordered parts of NS5A. NS5A-D2D3, despite its overall high flexibility, shows a large extent of local residual (α-helical and ß-turn) structure, as well as a network of electrostatic long-range interactions. Furthermore, we could demonstrate that these long-range interactions become modulated upon binding to the host protein Bin1, as well as after NS5A phosphorylation by CK2. As the charged peptide regions involved in these interactions are well conserved among the different HCV genotypes, these transient long-range interactions may be important for some of the functions of NS5A over the course of the HCV life cycle.

Conformational Polymorphism in Autophagy-Related Protein GATE-16.

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.