Human transferrin receptor can mediate SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been detected in almost all organs of coronavirus disease-19 patients, although some organs do not express angiotensin-converting enzyme-2 (ACE2), a known receptor of SARS-CoV-2, implying the presence of alternative receptors and/or co-receptors. Here, we show that the ubiquitously distributed human transferrin receptor (TfR), which binds to diferric transferrin to traffic between membrane and endosome for the iron delivery cycle, can ACE2-independently mediate SARS-CoV-2 infection. Human, not mouse TfR, interacts with Spike protein with a high affinity (KD ~2.95 nM) to mediate SARS-CoV-2 endocytosis. TfR knock-down (TfR-deficiency is lethal) and overexpression inhibit and promote SARS-CoV-2 infection, respectively. Humanized TfR expression enables SARS-CoV-2 infection in baby hamster kidney cells and C57 mice, which are known to be insusceptible to the virus infection. Soluble TfR, Tf, designed peptides blocking TfR-Spike interaction and anti-TfR antibody show significant anti-COVID-19 effects in cell and monkey models. Collectively, this report indicates that TfR is a receptor/co-receptor of SARS-CoV-2 mediating SARS-CoV-2 entry and infectivity by likely using the TfR trafficking pathway.

Orientational Nanoconjugation with Gold Endows Marked Antimicrobial Potential and Drugability of Ultrashort Dipeptides.

Antibiotic resistance is a global threat. Antimicrobial peptides (AMPs) are highly desirable to treat multidrug-resistant pathogen infection. However, few AMPs are clinically available, due to high cost, instability, and poor selectivity. Here, ultrashort AMPs (2-3 residues with an N-terminal cysteine) are designed and assembled as gold nanoparticles. Au-S conjugation and ultrashort size restrict nonspecific reactions and peptide orientation, thus concentrating positively charged residues on the surface. The nanostructured assemblies enormously enhance antimicrobial abilities by 1000-6000-fold and stability. One representative (Au-Cys-Arg-NH2, Au_CR) shows selective antibacterial activity against Staphylococcus aureus with 10 nM minimal inhibitory concentration. Au_CR has comparable or better in vivo antimicrobial potency than vancomycin and methicillin, with low propensity to induce resistance, little side effects, and high stability (17.5 h plasma half-life). Au_CR acts by inducing collapse of membrane potential and rupture of the bacterial membrane. The report provides insights for developing AMP-metal nanohybrids, particularly tethering nonspecific reactions and AMP orientation on the metal surface.

Design methods for antimicrobial peptides with improved performance.

The recalcitrance of pathogens to traditional antibiotics has made treating and eradicating bacterial infections more difficult. In this regard, developing new antimicrobial agents to combat antibiotic-resistant strains has become a top priority. Antimicrobial peptides (AMPs), a ubiquitous class of naturally occurring compounds with broad-spectrum antipathogenic activity, hold significant promise as an effective solution to the current antimicrobial resistance (AMR) crisis. Several AMPs have been identified and evaluated for their therapeutic application, with many already in the drug development pipeline. Their distinct properties, such as high target specificity, potency, and ability to bypass microbial resistance mechanisms, make AMPs a promising alternative to traditional antibiotics. Nonetheless, several challenges, such as high toxicity, lability to proteolytic degradation, low stability, poor pharmacokinetics, and high production costs, continue to hamper their clinical applicability. Therefore, recent research has focused on optimizing the properties of AMPs to improve their performance. By understanding the physicochemical properties of AMPs that correspond to their activity, such as amphipathicity, hydrophobicity, structural conformation, amino acid distribution, and composition, researchers can design AMPs with desired and improved performance. In this review, we highlight some of the key strategies used to optimize the performance of AMPs, including rational design and de novo synthesis. We also discuss the growing role of predictive computational tools, utilizing artificial intelligence and machine learning, in the design and synthesis of highly efficacious lead drug candidates.

Lipopolysaccharide-binding protein expression is increased by stress and inhibits monoamine synthesis to promote depressive symptoms.

Monoamine insufficiency is suggested to be associated with depressive features such as sadness, anhedonia, insomnia, and cognitive dysfunction, but the mechanisms that cause it are unclear. We found that the acute-phase protein lipopolysaccharide-binding protein (LBP) inhibits monoamine biosynthesis by acting as an endogenous inhibitor of dopamine-ß-hydroxylase (DBH) and aromatic-L-amino-acid-decarboxylase (DDC). LBP expression was increased in individuals with depression and by diverse stress challenges in mice. LBP antibodies and LBP knockdown inhibited monoamine insufficiency and depression-like features in mice, which worsened with LBP overexpression or administration. Monoamine insufficiency and depression-like symptoms were not induced by stressful stimuli in LBP-deficient mice, further highlighting a role for LBP in stress-induced depression, and a peptide we designed that blocks LBP-DBH and LBP-DDC interactions showed anti-depression effects in mice. This study reveals an important role for LBP in regulating monoamine biosynthesis and suggests that targeting LBP may have potential as a treatment for some individuals with depression.

Vertebrate OTOP1 is also an alkali-activated channel.

Although alkaline sensation is critical for survival, alkali-activated receptors are yet to be identified in vertebrates. Here, we showed that the OTOP1 channel can be directly activated by extracellular alkali. Notably, OTOP1 biphasically mediated proton influx and efflux with extracellular acid and base stimulation, respectively. Mutations of K221 and R554 at the S5-S6 and S11-S12 linkers significantly reduced alkali affinity without affecting acid activation, suggesting that different domains are responsible for acid- and alkali-activation of OTOP1. The selectivity for H+ was significantly higher in OTOP1 activated by alkali than that by acid, further suggesting that the two activations might be independent gating processes. Given that the alkali-activation of OTOP1 and the required key residues were conserved in the six representative vertebrates, we cautiously propose that OTOP1 participates in alkaline sensation in vertebrates. Thus, our study identified OTOP1 as an alkali-activated channel.

Human antimicrobial peptide LL-37 contributes to Alzheimer's disease progression.

As a prime mover in Alzheimer's disease (AD), microglial activation requires membrane translocation, integration, and activation of the metamorphic protein chloride intracellular channel 1 (CLIC1), which is primarily cytoplasmic under physiological conditions. However, the formation and activation mechanisms of functional CLIC1 are unknown. Here, we found that the human antimicrobial peptide (AMP) LL-37 promoted CLIC1 membrane translocation and integration. It also activates CLIC1 to cause microglial hyperactivation, neuroinflammation, and excitotoxicity. In mouse and monkey models, LL-37 caused significant pathological phenotypes linked to AD, including elevated amyloid-ß, increased neurofibrillary tangles, enhanced neuronal death and brain atrophy, enlargement of lateral ventricles, and impairment of synaptic plasticity and cognition, while Clic1 knockout and blockade of LL-37-CLIC1 interactions inhibited these phenotypes. Given AD's association with infection and that overloading AMP may exacerbate AD, this study suggests that LL-37, which is up-regulated upon infection, may be a driving force behind AD by acting as an endogenous agonist of CLIC1.

Hypoxia and low temperature upregulate transferrin to induce hypercoagulability at high altitude.

Studies have shown significantly increased thromboembolic events at high altitude. We recently reported that transferrin could potentiate blood coagulation, but the underlying mechanism for high altitude-related thromboembolism is still poorly understood. Here, we examined the activity and concentration of plasma coagulation factors and transferrin in plasma collected from long-term human residents and short-stay mice exposed to varying altitudes. We found that the activities of thrombin and factor XIIa (FXIIa) along with the concentrations of transferrin were significantly increased in the plasma of humans and mice at high altitudes. Furthermore, both hypoxia (6% O2) and low temperature (0°C), 2 critical high-altitude factors, enhanced hypoxia-inducible factor 1α (HIF-1α) levels to promote the expression of the transferrin gene, whose enhancer region contains HIF-1α binding site, and consequently, to induce hypercoagulability by potentiating thrombin and FXIIa. Importantly, thromboembolic disorders and pathological insults in mouse models induced by both hypoxia and low temperature were ameliorated by transferrin interferences, including transferrin antibody treatment, transferrin downregulation, and the administration of our designed peptides that inhibit the potentiation of transferrin on thrombin and FXIIa. Thus, low temperature and hypoxia upregulated transferrin expression-promoted hypercoagulability. Our data suggest that targeting the transferrin-coagulation pathway is a novel and potentially powerful strategy against thromboembolic events caused by harmful environmental factors under high-altitude conditions.

Bioactive Peptides and Proteins from Centipede Venoms.

Venoms are a complex cocktail of biologically active molecules, including peptides, proteins, polyamide, and enzymes widely produced by venomous organisms. Through long-term evolution, venomous animals have evolved highly specific and diversified peptides and proteins targeting key physiological elements, including the nervous, blood, and muscular systems. Centipedes are typical venomous arthropods that rely on their toxins primarily for predation and defense. Although centipede bites are frequently reported, the composition and effect of centipede venoms are far from known. With the development of molecular biology and structural biology, the research on centipede venoms, especially peptides and proteins, has been deepened. Therefore, we summarize partial progress on the exploration of the bioactive peptides and proteins in centipede venoms and their potential value in pharmacological research and new drug development.

Centipede Venom: A Potential Source of Ion Channel Modulators.

Centipedes are one of the most ancient and successful living venomous animals. They have evolved spooky venoms to deter predators or hunt prey, and are widely distributed throughout the world besides Antarctica. Neurotoxins are the most important virulence factor affecting the function of the nervous system. Ion channels and receptors expressed in the nervous system, including NaV, KV, CaV, and TRP families, are the major targets of peptide neurotoxins. Insight into the mechanism of neurotoxins acting on ion channels contributes to our understanding of the function of both channels and centipede venoms. Meanwhile, the novel structure and selective activities give them the enormous potential to be modified and exploited as research tools and biological drugs. Here, we review the centipede venom peptides that act on ion channels.

Venom resistance mechanisms in centipede show tissue specificity.

Venomous animals utilize venom glands to secrete and store powerful toxins for intraspecific and/or interspecific antagonistic interactions, implying that tissue-specific resistance is essential for venom glands to anatomically separate toxins from other tissues. Here, we show the mechanism of tissue-specific resistance in centipedes (Scolopendra subspinipes mutilans), where the splice variant of the receptor repels its own toxin. Unlike the well-known resistance mechanism by mutation in a given exon, we found that the KCNQ1 channel is highly expressed in the venom gland as a unique splice variant in which the pore domain and transmembrane domain six, partially encoded by exon 6 (rather than 7 as found in other tissues), contain eleven mutated residues. Such a splice variant is sufficient to gain resistance to SsTx (a lethal toxin for giant prey capture) in the venom gland due to a partially buried binding site. Therefore, the tissue-specific KCNQ1 modification confers resistance to the toxins, establishing a safe zone in the venom-storing/secreting environment.

Anticarin-ß shows a promising anti-osteosarcoma effect by specifically inhibiting CCT4 to impair proteostasis.

Unlike healthy, non-transformed cells, the proteostasis network of cancer cells is taxed to produce proteins involved in tumor development. Cancer cells have a higher dependency on molecular chaperones to maintain proteostasis. The chaperonin T-complex protein ring complex (TRiC) contains eight paralogous subunits (CCT1-8), and assists the folding of as many as 10% of cytosolic proteome. TRiC is essential for the progression of some cancers, but the roles of TRiC subunits in osteosarcoma remain to be explored. Here, we show that CCT4/TRiC is significantly correlated in human osteosarcoma, and plays a critical role in osteosarcoma cell survival. We identify a compound anticarin-ß that can specifically bind to and inhibit CCT4. Anticarin-ß shows higher selectivity in cancer cells than in normal cells. Mechanistically, anticarin-ß potently impedes CCT4-mediated STAT3 maturation. Anticarin-ß displays remarkable antitumor efficacy in orthotopic and patient-derived xenograft models of osteosarcoma. Collectively, our data uncover a key role of CCT4 in osteosarcoma, and propose a promising treatment strategy for osteosarcoma by disrupting CCT4 and proteostasis.

Potent CaV3.2 channel inhibitors exert analgesic effects in acute and chronic pain models.

Pain is the most common presenting physical symptom and a primary reason for seeking medical care, which chronically affects people's mental health and social life. CaV3.2 channel plays an essential role in the peripheral processing maintenance of pain states. This study was designed to identify novel drug candidates targeting the CaV3.2 channel. Whole-cell patch-clamp, cellular thermal shift assay, FlexStation, in vivo and in vitro CaV3.2 knock-down, site-directed mutagenesis, and double-mutant cycle analysis were employed to explore the pain-related receptors and ligand-receptor direct interaction. We found that toddaculin efficiently inhibits the CaV3.2 channel and significantly reduced the excitability of dorsal root ganglion neurons and pain behaviors. The Carbonyl group of coumarins directly interacts with the pore domain of CaV3.2 via van der Waals (VDW) force. Docking with binding pockets further led us to identify glycycoumarin, which exhibited more potent inhibition on the CaV3.2 channel and better analgesic activity than the parent compound. Toddaculin and its analog showed beneficial therapeutic effects in pain models. Toddaculin binding pocket on CaV3.2 might be a promising docking site for the design of drugs.

Bioactive Peptides and Proteins from Wasp Venoms.

Wasps, members of the order Hymenoptera, use their venom for predation and defense. Accordingly, their venoms contain various constituents acting on the circulatory, immune and nervous systems. Wasp venom possesses many allergens, enzymes, bioactive peptides, amino acids, biogenic amines, and volatile matters. In particular, some peptides show potent antimicrobial, anti-inflammatory, antitumor, and anticoagulant activity. Additionally, proteinous components from wasp venoms can cause tissue damage or allergic reactions in organisms. These bioactive peptides and proteins involved in wasp predation and defense may be potential sources of lead pharmaceutically active molecules. In this review, we focus on the advances in bioactive peptides and protein from the venom of wasps and their biological effects, as well as the allergic reactions and immunotherapy induced by the wasp venom.

An inhibitor of leukotriene-A4 hydrolase from bat salivary glands facilitates virus infection.

SignificanceAn immunosuppressant protein (MTX), which facilitates virus infection by inhibiting leukotriene A4 hydrolase (LTA4H) to produce the lipid chemoattractant leukotriene B4 (LTB4), was identified and characterized from the submandibular salivary glands of the bat Myotis pilosus. To the best of our knowledge, this is a report of an endogenous LTA4H inhibitor in animals. MTX was highly concentrated in the bat salivary glands, suggesting a mechanism for the generation of immunological privilege and immune tolerance and providing evidence of viral shedding through oral secretions. Moreover, given that the immunosuppressant MTX selectively inhibited the proinflammatory activity of LTA4H, without affecting its antiinflammatory activity, MTX might be a potential candidate for the development of antiinflammatory drugs by targeting the LTA4-LTA4H-LTB4 inflammatory axis.

Shrew's venom quickly causes circulation disorder, analgesia and hypokinesia.

Multiple representatives of eulipotyphlan mammals such as shrews have oral venom systems. Venom facilitates shrews to hunt and/or hoard preys. However, little is known about their venom composition, and especially the mechanism to hoard prey in comatose states for meeting their extremely high metabolic rates. A toxin (BQTX) was identified from venomous submaxillary glands of the shrew Blarinella quadraticauda. BQTX is specifically distributed and highly concentrated (~ 1% total protein) in the organs. BQTX shares structural and functional similarities to toxins from snakes, wasps and snails, suggesting an evolutional relevancy of venoms from mammalians and non-mammalians. By potentiating thrombin and factor-XIIa and inhibiting plasmin, BQTX induces acute hypertension, blood coagulation and hypokinesia. It also shows strong analgesic function by inhibiting elastase. Notably, the toxin keeps high plasma stability with a 16-h half-life in-vivo, which likely extends intoxication to paralyze or immobilize prey hoarded fresh for later consumption and maximize foraging profit.

The role of TRPA1 channels in thermosensation.

Transient receptor potential ankyrin 1 (TRPA1) is a polymodal nonselective cation channel sensitive to different physical and chemical stimuli. TRPA1 is associated with many important physiological functions in different species and thus is involved in different degrees of evolution. TRPA1 acts as a polymodal receptor for the perceiving of irritating chemicals, cold, heat, and mechanical sensations in various animal species. Numerous studies have supported many functions of TRPA1, but its temperature-sensing function remains controversial. Although TRPA1 is widely distributed in both invertebrates and vertebrates, and plays a crucial role in tempreture sensing, the role of TRPA1 thermosensation and molecular temperature sensitivity are species-specific. In this review, we summarize the temperature-sensing role of TRPA1 orthologues in terms of molecular, cellular, and behavioural levels.

Peptide OPTX-1 From Ornithodoros papillipes Tick Inhibits the pS273R Protease of African Swine Fever Virus.

African swine fever virus (ASFV) is a large double-stranded DNA virus and causes high mortality in swine. ASFV can be transmitted by biological vectors, including soft ticks in genus Ornithodoros but not hard ticks. However, the underlying mechanisms evolved in the vectorial capacity of soft ticks are not well-understood. Here, we found that a defensin-like peptide toxin OPTX-1 identified from Ornithodoros papillipes inhibits the enzyme activity of the ASFV pS273R protease with a Ki =0.821±0.526µM and shows inhibitory activity on the replication of ASFV. The analogs of OPTX-1 from hard ticks show more inhibitory efficient on pS273R protease. Considering that ticks are blood-sucking animals, we tested the effects of OPTX-1 and its analogs on the coagulation system. At last, top 3D structures represented surface analyses of the binding sites of pS273R with different inhibitors that were obtained by molecular docking based on known structural information. In summary, our study provides evidence that different inhibitory efficiencies between soft tick-derived OPTX-1 and hard tick-derived defensin-like peptides may determine the vector and reservoir competence of ticks.

Toward an understanding of tree frog (Hyla japonica) for predator deterrence.

Gene-encoded peptides with distinct potent bioactivities enable several animals to take advantage of fierce interspecific interaction, as seen in the skin secretion of amphibians. Unlike, most amphibian species that frequently switches terrestrial-aquatic habitats and hides easily from terrestrial predators, tree frogs of small body size are considered as the vulnerable prey in the arboreal habitat. Here, we show the structural and functional diversity of peptide families based on the skin transcriptome of Hyla japonica, which has evolved to be wrapped as an efficient chemical toolkit for defensive use in arboreal habitat. Generally, the presence of antimicrobial peptide and proteinase inhibitor families reveals the functional consistency of Hyla japonica skin compared to other amphibian species. Furthermore, we found that Anntoxin-like neurotoxins with high expression levels are species-specific in tree frogs. Interestingly, derivatives in the Anntoxin-like family exhibit multiple evolutionary traits in modifying the copy number, folding type, and three-dimensional architecture, which are considered essential for targeting the ion channels of terrestrial predators. Together, our study not only reveals the peptide diversity in the skin secretion of H. japonica, but also draws insights into the predator-deterring strategy for coping with arboreal habitat.

A plant-derived TRPV3 inhibitor suppresses pain and itch.

BACKGROUND AND PURPOSE: Itching is the most frequent pathology in dermatology that has significant impacts on people's mental health and social life. Transient receptor potential vanilloid 3 (TRPV3) channel is a promising target for treating pruritus. However, few selecetive and potent antagonists have been reported. This study was designed to identify selective TRPV3 antagonist and elucidate its anti-pruritus pharmacology. EXPERIMENTAL APPROACH: FlexStation and calcium fluorescence imaging were conducted to track the functional compounds. Whole-cell patch clamp was used to record itch-related ion channel currents. Homologous recombination and site-directed mutagenesis were employed to construct TRPV3 channel chimeras and point mutations for exploring pharmacological mechanism. Mouse models were used for in vivo anti-pruritus assay. KEY RESULTS: An acridone alkaloid (citrusinine-II) was purified and characterized from Atalantia monophylla. It directly interacts with Y564 within S4 helix of TRPV3 to selectively inhibit the channel with a half maximal inhibitory concentration (IC50 ) of 12.43 µM. Citrusinine-II showed potential efficacy to attenuate both chronic and acute itch. Intradermal administration of citrusinine-II (143 ng/skin site) nearly completely inhibited itch behaviours. It also shows significant analgesic effects. Little side effects of the compound are observed. CONCLUSION AND IMPLICATIONS: By acting as a selective and potent inhibitor of TRPV3 channel, citrusinine-II shows valuable therapeutic effects in pruritus animal models and is a promising candidate drug and/or lead molecule for the development of anti-pruritus drugs.