RNA-binding protein-regulated fibronectin is essential for EGFR-activated metastasis of head and neck squamous cell carcinoma.

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

ToxMPNN: A deep learning model for small molecule toxicity prediction.

Machine learning (ML) has shown a great promise in predicting toxicity of small molecules. However, the availability of data for such predictions is often limited. Because of the unsatisfactory performance of models trained on a single toxicity endpoint, we collected toxic small molecules with multiple toxicity endpoints from previous study. The dataset comprises 27 toxic endpoints categorized into seven toxicity classes, namely, carcinogenicity and mutagenicity, acute oral toxicity, respiratory toxicity, irritation and corrosion, cardiotoxicity, CYP450, and endocrine disruption. In addition, a binary classification Common-Toxicity task was added based on the aforementioned dataset. To improve the performance of the models, we added marketed drugs as negative samples. This study presents a toxicity predictive model, ToxMPNN, based on the message passing neural network (MPNN) architecture, aiming to predict the toxicity of small molecules. The results demonstrate that ToxMPNN outperforms other models in capturing toxic features within the molecular structure, resulting in more precise predictions with the ROC_AUC testing score of 0.886 for the Toxicity_drug dataset. Furthermore, it was observed that adding marketed drugs as negative samples not only improves the predictive performance of the binary classification Common-Toxicity task but also enhances the stability of the model prediction. It shows that the graph-based deep learning (DL) algorithms in this study can be used as a trustworthy and effective tool to assess small molecule toxicity in the development of new drugs.

Small Open Reading Frame-Encoded Micro-Peptides: An Emerging Protein World.

Small open reading frames (sORFs) are often overlooked features in genomes. In the past, they were labeled as noncoding or "transcriptional noise". However, accumulating evidence from recent years suggests that sORFs may be transcribed and translated to produce sORF-encoded polypeptides (SEPs) with less than 100 amino acids. The vigorous development of computational algorithms, ribosome profiling, and peptidome has facilitated the prediction and identification of many new SEPs. These SEPs were revealed to be involved in a wide range of basic biological processes, such as gene expression regulation, embryonic development, cellular metabolism, inflammation, and even carcinogenesis. To effectively understand the potential biological functions of SEPs, we discuss the history and development of the newly emerging research on sORFs and SEPs. In particular, we review a range of recently discovered bioinformatics tools for identifying, predicting, and validating SEPs as well as a variety of biochemical experiments for characterizing SEP functions. Lastly, this review underlines the challenges and future directions in identifying and validating sORFs and their encoded micropeptides, providing a significant reference for upcoming research on sORF-encoded peptides.

Molecular mechanisms of centipede toxin SsTx-4 inhibition of inwardly rectifying potassium channels.

Inwardly rectifying potassium channels (Kirs) are important drug targets, with antagonists for the Kir1.1, Kir4.1, and pancreatic Kir6.2/SUR1 channels being potential drug candidates for treating hypertension, depression, and diabetes, respectively. However, few peptide toxins acting on Kirs are identified and their interacting mechanisms remain largely elusive yet. Herein, we showed that the centipede toxin SsTx-4 potently inhibited the Kir1.1, Kir4.1, and Kir6.2/SUR1 channels with nanomolar to submicromolar affinities and intensively studied the molecular bases for toxin-channel interactions using patch-clamp analysis and site-directed mutations. Other Kirs including Kir2.1 to 2.4, Kir4.2, and Kir7.1 were resistant to SsTx-4 treatment. Moreover, SsTx-4 inhibited the inward and outward currents of Kirs with different potencies, possibly caused by a K+ "knock-off" effect, suggesting the toxin functions as an out pore blocker physically occluding the K+-conducting pathway. This conclusion was further supported by a mutation analysis showing that M137 located in the outer vestibule of the Kir6.2/ΔC26 channel was the key residue mediating interaction with SsTx-4. On the other hand, the molecular determinants within SsTx-4 for binding these Kir channels only partially overlapped, with K13 and F44 being the common key residues. Most importantly, K11A, P15A, and Y16A mutant toxins showed improved affinity and/or selectivity toward Kir6.2, while R12A mutant toxin had increased affinity for Kir4.1. To our knowledge, SsTx-4 is the first characterized peptide toxin with Kir4.1 inhibitory activity. This study provides useful insights for engineering a Kir6.2/SUR1 channel-specific antagonist based on the SsTx-4 template molecule and may be useful in developing new antidiabetic drugs.

Engineering of highly potent and selective HNTX-III mutant against hNav1.7 sodium channel for treatment of pain.

Human voltage-gated sodium channel Nav1.7 (hNav1.7) is involved in the generation and conduction of neuropathic and nociceptive pain signals. Compelling genetic and preclinical studies have validated that hNav1.7 is a therapeutic target for the treatment of pain; however, there is a dearth of currently available compounds capable of targeting hNav1.7 with high potency and specificity. Hainantoxin-III (HNTX-III) is a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. Here, we report the engineering of improved potency and Nav selectivity of hNav1.7 inhibition peptides derived from the HNTX-III scaffold. Alanine scanning mutagenesis showed key residues for HNTX-III interacting with hNav1.7. Site-directed mutagenesis analysis indicated key residues on hNav1.7 interacting with HNTX-III. Molecular docking was conducted to clarify the binding interface between HNTX-III and Nav1.7 and guide the molecular engineering process. Ultimately, we obtained H4 [K0G1-P18K-A21L-V] based on molecular docking of HNTX-III and hNav1.7 with a 30-fold improved potency (IC50 0.007 ± 0.001 µM) and >1000-fold selectivity against Nav1.4 and Nav1.5. H4 also showed robust analgesia in the acute and chronic inflammatory pain model and neuropathic pain model. Thus, our results provide further insight into peptide toxins that may prove useful in guiding the development of inhibitors with improved potency and selectivity for Nav subtypes with robust analgesia.

Naja atra venom peptide reduces pain by selectively blocking the voltage-gated sodium channel Nav1.8.

The voltage-gated sodium channel Nav1.8 is preferentially expressed in peripheral nociceptive neurons and contributes to inflammatory and neuropathic pain. Therefore, Nav1.8 has emerged as one of the most promising analgesic targets for pain relief. Using large-scale screening of various animal-derived toxins and venoms for Nav1.8 inhibitors, here we identified µ-EPTX-Na1a, a 62-residue three-finger peptide from the venom of the Chinese cobra (Naja atra), as a potent inhibitor of Nav1.8, exhibiting high selectivity over other voltage-gated sodium channel subtypes. Using whole-cell voltage-clamp recordings, we observed that purified µ-EPTX-Na1a blocked the Nav1.8 current. This blockade was associated with a depolarizing shift of activation and repolarizing shift of inactivation, a mechanism distinct from that of any other gating modifier toxin identified to date. In rodent models of inflammatory and neuropathic pain, µ-EPTX-Na1a alleviated nociceptive behaviors more potently than did morphine, indicating that µ-EPTX-Na1a has a potent analgesic effect. µ-EPTX-Na1a displayed no evident cytotoxicity and cardiotoxicity and produced no obvious adverse responses in mice even at a dose 30-fold higher than that producing a significant analgesic effect. Our study establishes µ-EPTX-Na1a as a promising lead for the development of Nav1.8-targeting analgesics to manage pain.

Drug-Bearing Peptide-Based Nanospheres for the Inhibition of Metastasis and Growth of Cancer.

Employing a peptide-based nanoscale drug delivery system is an effective strategy to overcome the poor therapeutic outcomes of chemotherapeutic drugs. Here, we developed a self-assembling peptide-drug delivery system comprising a self-assembling anticancer peptide (R-lycosin-I), as revealed in our previous study, and 10-hydroxycamptothecin (HCPT) for cancer therapy. The results showed that peptide-drug conjugates (R-L-HCPT) could assemble into nanospheres of 40-60 nm in water. Compared with free HCPT, R-L-HCPT nanospheres not only inhibited tumor growth but also suppressed pulmonary metastatic nodules on B16-F10 cells in vivo. In summary, these results indicated that the self-assembling R-lycosin-I could provide a promising nanoscale platform for delivering small-molecule drugs. Moreover, our study might provide new opportunities for the development of a new class of functional peptide-drug-conjugated systems based on nanomaterials, which could synergistically enhance anticancer outcomes.

Molecular diversification of antimicrobial peptides from the wolf spider Lycosa sinensis venom based on peptidomic, transcriptomic, and bioinformatic analyses.

The venom of Lycosoidea spiders is a complex multicomponent mixture of neurotoxic peptides (main components) and antimicrobial peptides (AMPs) as minor components. In this study, we described the high-throughput identification and analysis of AMPs from Lycosa sinensis venom (named LS-AMPs) using a combination strategy that includes the following three different analysis approaches: (i) peptidomic analysis, namely reversed-phase high-performance liquid chromatography (RP-HPLC) separation plus top-down sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS); (ii) transcriptomic analysis, namely cDNA library construction plus DNA sequencing; (iii) bioinformatic analysis, namely analysis and prediction for molecular characters of LS-AMPs by the online biology databases. In total, 52 sequences of AMPs were identified from L. sinensis venom, and all AMPs can be categorized into eight different families according to phylogenetic analysis and sequence identity. This is the largest number of AMPs identified from a spider species so far. In the present study, we demonstrated molecular characteristics, such as complex precursor, N- and/or C-terminally truncated analogs, and C-terminal amidation of LS-AMPs from L. sinensis venom. This is a preliminary investigation on the molecular diversification of venom-derived AMPs from the wolf spider species (family Lycosidae), and a detailed investigation on the functional diversity of LS-AMPs will be preformed in the future.

Structural Foundation for Insect-Selective Activity of Acylpolyamine Toxins from Spider Araneus ventricosus.

Spider venoms are insecticidal mixtures with diverse biological activities, and acylpolyamines are their small molecular active components. However, the mechanism for the insecticidal activity of acylpolyamines remains to be elucidated. Here, the structure and function of two acylpolyamine toxins, AVTX-622 and AVTX-636, from Araneus ventricosus were investigated. Nuclear magnetic resonance (NMR) analysis illustrated that the structure of two toxins was very similar, and compared to AVTX-636, AVTX-622 only missed a methylene group in the linker region between the polyamine head and tail. Both the two toxins did not inhibit on voltage-gated sodium channels in mammalian neuronal cells. Intriguingly, AVTX-622, but not AVTX-636, inhibited voltage-gated sodium channels in DUM neuronal cells of Periplaneta americana. Further animal test displayed that the paralyzing potency of AVTX-622 on insect was over ten-times stronger than that of AVTX-636. These findings indicate that a single methylene deletion from AVTX-636 offered AVTX-622 the insect-selective voltage-gated sodium channel activity, which not only elucidated structure-function of the toxins, but also provided new clues for insect-selective insecticide design.

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

Voltage-gated sodium channels (NaVs) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial NaVs have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial NaVs. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula Chilobrachys jingzhao as a high-affinity antagonist of the prokaryotic NaVs NsVBa (nonselective voltage-gated Bacillus alcalophilus) and NaChBac (bacterial sodium channel from Bacillus halodurans) (IC50 = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of NsVBa, whereas the local anesthetic drug lidocaine was shown to antagonize NsVBa without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of NsVBa, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and in silico analysis suggested that JZTx-27 trapped VSM of NsVBa in one of the deactivated states. In mammalian NaVs, JZTx-27 preferably inhibited the inactivation of NaV1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic NaVs. More important, we proposed that JZTx-27 stabilized the NsVBa VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of NaVs.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

The Roles of Fatty-Acid Modification in the Activity of the Anticancer Peptide R-Lycosin-I.

We previously reported that R-lycosin-I, modified by amino acid substitution from lycosin-I, was a peptide with anticancer activity and a linear amphipathic α-helix conformation and that it can induce cancer cell apoptosis and inhibit cell proliferation. However, the anticancer activity of R-lycosin-I was not highly improved. In order to further improve the anticancer activity of R-lycosin-I, fatty acids with different chain lengths from 12 to 20 carbons were introduced to the N-terminal of R-lycosin-I to yield five lipopeptides (R-C12, R-C14, R-C16, R-C18, R-C20). The physicochemical properties of the five lipopeptides were determined by hydrodynamic size, ζ-potential, and circular dichroism spectroscopy, respectively. Then, the cytotoxic activity of these lipopeptides in A549 cells was evaluated with serum-containing and serum-free media, respectively, showing their anticancer activities were all increased through fatty-acid modification. This may be a result of the increased hydrophobicity and the enhanced interaction with the cancer cell membrane. The cytotoxic activity of R-C16 was 3-4-fold higher than that of the original R-lycosin-I and also was the strongest among all five lipopeptides, whether in serum or serum-free conditions. Compared with R-lycosin-I, the lactate dehydrogenase (LDH) leakage assay and scanning electron microscopy (SEM) indicated that R-C16 had a weakly destructive effect on the cancer cell membrane, but it might cause apoptosis to exert an anticancer activity. Finally, the impacts of fatty-acid length on the physicochemical properties and the anticancer potential of peptide were discussed. Our data consolidate work on fatty-acid-modified anticancer peptides.

Arginine modification of lycosin-I to improve inhibitory activity against cancer cells.

Lycosin-I is a linear amphipathic α-helical anticancer peptide (ACP) extracted from the spider Lycosa singoriensis, which can activate the mitochondrial death pathway to induce apoptosis in tumor cells and up-regulate p27 to inhibit cell proliferation. However, the applicability of lycosin-I as a novel anticancer drug is limited by its low cellular entry and efficacy in solid tumors. Amino acid substitution presents an effective and modest strategy to improve the anticancer activity and bioavailability of ACPs. Herein, an arginine-modified lycosin-I (named R-lycosin-I) was designed and synthesized by substituting lysine (Lys) with arginine (Arg). This peptide exhibited higher anticancer activity and penetrability against solid tumor cells than lycosin-I. They displayed noticeable differences in their physicochemical properties including the secondary structure, hydrodynamic size, and zeta potential. Fluorescence analyses have confirmed that R-lycosin-I exhibits increased cellular uptake and improved intracellular distribution. Due to its superior physical and chemical properties and high serum stability, R-lycosin-I could penetrate deeply into tumor spheroids and produce strong toxicity in the 3D tumor model. Overall, these findings suggest that arginine modification may provide an effective strategy for improving the anticancer activity of lycosin-I, and R-lycosin-I may be a useful lead for developing anticancer drugs.

Structural and Functional Diversity of Peptide Toxins from Tarantula Haplopelma hainanum (Ornithoctonus hainana) Venom Revealed by Transcriptomic, Peptidomic, and Patch Clamp Approaches.

Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.

Rat liver sinusoidal surface N-linked glycoproteomic analysis by affinity enrichment and mass spectrometric identification.

Glycosylation in liver is one of the most biologically important protein modifications. It plays critical roles in many physiological and pathological processes by virtue of its unique location at the blood-tissue interface, including angiogenesis, liver cancer, cirrhosis, and fibrosis. To analyze glycosylation of plasma membrane proteins in liver sinusoidal endothelial cells (LSEC), N-glycopeptides of the LSEC surface were enriched using a filter-assisted sample preparation-based lectin affinity capture method and subsequently identified with mass spectrometry. In total, 225 unique N-glycosylation sites on 152 glycoproteins were identified, of which 119 (53%) sites had not previously been determined experimentally. Among the glycoproteins, 53% were classified as plasma membrane proteins and 47 (31%) as signaling proteins and receptors. Moreover, 23 cluster of differentiation antigens with 49 glycopeptides were detected within the membrane glycoproteins of the liver sinusoidal surface. Furthermore, bioinformatics analysis revealed that the majority of identified glycoproteins have an impact on processes of LSEC. Therefore, N-glycoproteomic analysis of the liver sinusoidal surface may provide useful information on liver regeneration and facilitate liver disease diagnosis.

Structure and function of hainantoxin-III, a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels isolated from the Chinese bird spider Ornithoctonus hainana.

In the present study, we investigated the structure and function of hainantoxin-III (HNTX-III), a 33-residue polypeptide from the venom of the spider Ornithoctonus hainana. It is a selective antagonist of neuronal tetrodotoxin-sensitive voltage-gated sodium channels. HNTX-III suppressed Nav1.7 current amplitude without significantly altering the activation, inactivation, and repriming kinetics. Short extreme depolarizations partially activated the toxin-bound channel, indicating voltage-dependent inhibition of HNTX-III. HNTX-III increased the deactivation of the Nav1.7 current after extreme depolarizations. The HNTX-III·Nav1.7 complex was gradually dissociated upon prolonged strong depolarizations in a voltage-dependent manner, and the unbound toxin rebound to Nav1.7 after a long repolarization. Moreover, analysis of chimeric channels showed that the DIIS3-S4 linker was critical for HNTX-III binding to Nav1.7. These data are consistent with HNTX-III interacting with Nav1.7 site 4 and trapping the domain II voltage sensor in the closed state. The solution structure of HNTX-III was determined by two-dimensional NMR and shown to possess an inhibitor cystine knot motif. Structural analysis indicated that certain basic, hydrophobic, and aromatic residues mainly localized in the C terminus may constitute an amphiphilic surface potentially involved in HNTX-III binding to Nav1.7. Taken together, our results show that HNTX-III is distinct from ß-scorpion toxins and other ß-spider toxins in its mechanism of action and binding specificity and affinity. The present findings contribute to our understanding of the mechanism of toxin-sodium channel interaction and provide a useful tool for the investigation of the structure and function of sodium channel isoforms and for the development of analgesics.

Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism.

Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca(2+)-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes.

Physiological and biochemical analysis to reveal the molecular basis for black widow spiderling toxicity.

The early research found that the spiderlings of black widow spider (Latrodectus tredecimguttatus) exhibited obvious toxicity to animals. The present work performed a systematical analysis of the aqueous extract of newborn black widow spiderlings. The extract was shown to contain 69.42% of proteins varying in molecular weights and isoelectric points. Abdominal injection of the extract into mice and cockroaches caused obvious poisoning symptoms as well as death, with LD50 being 5.30 mg/kg in mice and 16.74 µg/g in Periplaneta americana. Electrophysiological experiments indicated that the extract at a concentration of 10 µg/mL could completely block the neuromuscular transmission in isolated mouse nerve-hemidiaphragm preparations within 21 ± 1.5 min, and 100 µg/mL extract could inhibit a certain percentage of voltage-activated Na⁺, K⁺, and Ca²âº channel currents in rat dorsal root ganglion neurons. These results demonstrate that the spiderlings are rich in neurotoxic components, which play important roles in the spiderling toxicity.

Chemical punch packed in venoms makes centipedes excellent predators.

Centipedes are excellent predatory arthropods that inject venom to kill or immobilize their prey. Although centipedes have long been known to be venomous, their venoms remain largely unexplored. The chemical components responsible for centipede predation and the functional mechanisms are unknown. Twenty-six neurotoxin-like peptides belonging to ten groups were identified from the centipede venoms, Scolopendra subspinipes mutilans L. Koch by peptidomics combined with transcriptome analysis, revealing the diversity of neurotoxins. These neurotoxins each contain two to four intramolecular disulfide bridges, and in most cases the disulfide framework is different from that found in neurotoxins from the venoms of spiders, scorpions, marine cone snails, sea anemones, and snakes (5S animals). Several neurotoxins contain potential insecticidal abilities, and they are found to act on voltage-gated sodium, potassium, and calcium channels, respectively. Although these neurotoxins are functionally similar to the disulfide-rich neurotoxins found in the venoms of 5S animals in that they modulate the activity of voltage-gated ion channels, in almost all cases the primary structures of the centipede venom peptides are unique. This represents an interesting case of convergent evolution in which different venomous animals have evolved different molecular strategies for targeting the same ion channels in prey and predators. Moreover, the high level of biochemical diversity revealed in this study suggests that centipede venoms might be attractive subjects for prospecting and screening for peptide candidates with potential pharmaceutical or agrochemical applications.

Targeting the PDK/PDH axis to reverse metabolic abnormalities by structure-based virtual screening with in vitro and in vivo experiments.

In humans and animals, the pyruvate dehydrogenase kinase (PDK) family proteins (PDKs 1-4) are excessively activated in metabolic disorders such as obesity, diabetes, and cancer, inhibiting the activity of pyruvate dehydrogenase (PDH) which plays a crucial role in energy and fatty acid metabolism and impairing its function. Intervention and regulation of PDH activity have become important research approaches for the treatment of various metabolic disorders. In this study, a small molecule (g25) targeting PDKs and activating PDH, was identified through multi-level computational screening methods. In vivo and in vitro experiments have shown that g25 activated the activity of PDH and reduced plasma lactate and triglyceride level. Besides, g25 significantly decreased hepatic fat deposition in a diet-induced obesity mouse model. Furthermore, g25 enhanced the tumor-inhibiting activity of cisplatin when used in combination. Molecular dynamics simulations and in vitro kinase assay also revealed the specificity of g25 towards PDK2. Overall, these findings emphasize the importance of targeting the PDK/PDH axis to regulate PDH enzyme activity in the treatment of metabolic disorders, providing directions for future related research. This study provides a possible lead compound for the PDK/PDH axis related diseases and offers insights into the regulatory mechanisms of this pathway in diseases.