Sensory nerve regulation of bone homeostasis: Emerging therapeutic opportunities for bone-related diseases.

Understanding the intricate interplay between sensory nerves and bone tissue cells is of paramount significance in the field of bone biology and clinical medicine. The regulatory role of sensory nerves in bone homeostasis offers a novel perspective for the development of targeted therapeutic interventions for a spectrum of bone-related diseases, including osteoarthritis, osteoporosis, and intervertebral disc degeneration. By elucidating the mechanisms through which sensory nerves and their neuropeptides influence the differentiation and function of bone tissue cells, this review aims to shed light on emerging therapeutic targets that harness the neuro-skeletal axis for the treatment and management of debilitating bone disorders. Moreover, a comprehensive understanding of sensory nerve-mediated bone regulation may pave the way for the development of innovative strategies to promote bone health and mitigate the burden of skeletal pathologies in clinical practice.

The bone-liver interaction modulates immune and hematopoietic function through Pinch-Cxcl12-Mbl2 pathway.

Mesenchymal stromal cells (MSCs) are used to treat infectious and immune diseases and disorders; however, its mechanism(s) remain incompletely defined. Here we find that bone marrow stromal cells (BMSCs) lacking Pinch1/2 proteins display dramatically reduced ability to suppress lipopolysaccharide (LPS)-induced acute lung injury and dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice. Prx1-Cre; Pinch1f/f; Pinch2-/- transgenic mice have severe defects in both immune and hematopoietic functions, resulting in premature death, which can be restored by intravenous injection of wild-type BMSCs. Single cell sequencing analyses reveal dramatic alterations in subpopulations of the BMSCs in Pinch mutant mice. Pinch loss in Prx1+ cells blocks differentiation and maturation of hematopoietic cells in the bone marrow and increases production of pro-inflammatory cytokines TNF-α and IL-1ß in monocytes. We find that Pinch is critical for expression of Cxcl12 in BMSCs; reduced production of Cxcl12 protein from Pinch-deficient BMSCs reduces expression of the Mbl2 complement in hepatocytes, thus impairing the innate immunity and thereby contributing to infection and death. Administration of recombinant Mbl2 protein restores the lethality induced by Pinch loss in mice. Collectively, we demonstrate that the novel Pinch-Cxcl12-Mbl2 signaling pathway promotes the interactions between bone and liver to modulate immunity and hematopoiesis and may provide a useful therapeutic target for immune and infectious diseases.

Targeting Kindlin-2 in adipocytes increases bone mass through inhibiting FAS/PPARγ/FABP4 signaling in mice.

Osteoporosis (OP) is a systemic skeletal disease that primarily affects the elderly population, which greatly increases the risk of fractures. Here we report that Kindlin-2 expression in adipose tissue increases during aging and high-fat diet fed and is accompanied by decreased bone mass. Kindlin-2 specific deletion (K2KO) controlled by Adipoq-Cre mice or adipose tissue-targeting AAV (AAV-Rec2-CasRx-sgK2) significantly increases bone mass. Mechanistically, Kindlin-2 promotes peroxisome proliferator-activated receptor gamma (PPARγ) activation and downstream fatty acid binding protein 4 (FABP4) expression through stabilizing fatty acid synthase (FAS), and increased FABP4 inhibits insulin expression and decreases bone mass. Kindlin-2 inhibition results in accelerated FAS degradation, decreased PPARγ activation and FABP4 expression, and therefore increased insulin expression and bone mass. Interestingly, we find that FABP4 is increased while insulin is decreased in serum of OP patients. Increased FABP4 expression through PPARγ activation by rosiglitazone reverses the high bone mass phenotype of K2KO mice. Inhibition of FAS by C75 phenocopies the high bone mass phenotype of K2KO mice. Collectively, our study establishes a novel Kindlin-2/FAS/PPARγ/FABP4/insulin axis in adipose tissue modulating bone mass and strongly indicates that FAS and Kindlin-2 are new potential targets and C75 or AAV-Rec2-CasRx-sgK2 treatment are potential strategies for OP treatment.

The role of E3 ubiquitin ligases in bone homeostasis and related diseases.

The ubiquitin-proteasome system (UPS) dedicates to degrade intracellular proteins to modulate demic homeostasis and functions of organisms. These enzymatic cascades mark and modifies target proteins diversly through covalently binding ubiquitin molecules. In the UPS, E3 ubiquitin ligases are the crucial constituents by the advantage of recognizing and presenting proteins to proteasomes for proteolysis. As the major regulators of protein homeostasis, E3 ligases are indispensable to proper cell manners in diverse systems, and they are well described in physiological bone growth and bone metabolism. Pathologically, classic bone-related diseases such as metabolic bone diseases, arthritis, bone neoplasms and bone metastasis of the tumor, etc., were also depicted in a UPS-dependent manner. Therefore, skeletal system is versatilely regulated by UPS and it is worthy to summarize the underlying mechanism. Furthermore, based on the current status of treatment, normal or pathological osteogenesis and tumorigenesis elaborated in this review highlight the clinical significance of UPS research. As a strategy possibly remedies the limitations of UPS treatment, emerging PROTAC was described comprehensively to illustrate its potential in clinical application. Altogether, the purpose of this review aims to provide more evidence for exploiting novel therapeutic strategies based on UPS for bone associated diseases.

Talin-1 inhibits Smurf1-mediated Stat3 degradation to modulate ß-cell proliferation and mass in mice.

Insufficient pancreatic ß-cell mass and reduced insulin expression are key events in the pathogenesis of diabetes mellitus (DM). Here we demonstrate the high expression of Talin-1 in ß-cells and that deficiency of Talin-1 reduces ß-cell proliferation, which leads to reduced ß-cell mass and insulin expression, thus causing glucose intolerance without affecting peripheral insulin sensitivity in mice. High-fat diet fed exerbates these phenotypes. Mechanistically, Talin-1 interacts with the E3 ligase smad ubiquitination regulatory factor 1 (Smurf1), which prohibits ubiquitination of the signal transducer and activator of transcription 3 (Stat3) mediated by Smurf1, and ablation of Talin-1 enhances Smurf1-mediated ubiquitination of Stat3, leading to decreased ß-cell proliferation and mass. Furthermore, haploinsufficiency of Talin-1 and Stat3 genes, but not that of either gene, in ß-cell in mice significantly impairs glucose tolerance and insulin expression, indicating that both factors indeed function in the same genetic pathway. Finally, inducible deletion Talin-1 in ß-cell causes glucose intolerance in adult mice. Collectively, our findings reveal that Talin-1 functions as a crucial regulator of ß-cell mass, and highlight its potential as a therapeutic target for DM patients.

Kindlin-2 controls angiogenesis through modulating Notch1 signaling.

Kindlin-2 is critical for development and homeostasis of key organs, including skeleton, liver, islet, etc., yet its role in modulating angiogenesis is unknown. Here, we report that sufficient KINDLIN-2 is extremely important for NOTCH-mediated physiological angiogenesis. The expression of KINDLIN-2 in HUVECs is significantly modulated by angiogenic factors such as vascular endothelial growth factor A or tumor necrosis factor α. A strong co-localization of CD31 and Kindlin-2 in tissue sections is demonstrated by immunofluorescence staining. Endothelial-cell-specific Kindlin-2 deletion embryos die on E10.5 due to hemorrhage caused by the impaired physiological angiogenesis. Experiments in vitro show that vascular endothelial growth factor A-induced multiple functions of endothelial cells, including migration, matrix proteolysis, morphogenesis and sprouting, are all strengthened by KINDLIN-2 overexpression and severely impaired in the absence of KINDLIN-2. Mechanistically, we demonstrate that KINDLIN-2 inhibits the release of Notch intracellular domain through binding to and maintaining the integrity of NOTCH1. The impaired angiogenesis and avascular retinas caused by KINDLIN-2 deficiency can be rescued by DAPT, an inhibitor of γ-secretase which releases the intracellular domain from NOTCH1. Moreover, we demonstrate that high glucose stimulated hyperactive angiogenesis by increasing KINDLIN-2 expression could be prevented by KINDLIN-2 knockdown, indicating Kindlin-2 as a potential therapeutic target in treatment of diabetic retinopathy. Our study for the first time demonstrates the significance of Kindlin-2 in determining Notch-mediated angiogenesis during development and highlights Kindlin-2 as the potential therapeutic target in angiogenic diseases, such as diabetic retinopathy.

A novel sprayable thermosensitive hydrogel coupled with zinc modified metformin promotes the healing of skin wound.

A novel sprayable adhesive is established (ZnMet-PF127) by the combination of a thermosensitive hydrogel (Pluronic F127, PF127) and a coordination complex of zinc and metformin (ZnMet). Here we demonstrate that ZnMet-PF127 potently promotes the healing of traumatic skin defect and burn skin injury by promoting cell proliferation, angiogenesis, collagen formation. Furthermore, we find that ZnMet could inhibit reactive oxygen species (ROS) production through activation of autophagy, thereby protecting cell from oxidative stress induced damage and promoting healing of skin wound. ZnMet complex exerts better effects on promoting skin wound healing than ZnCl2 or metformin alone. ZnMet complex also displays excellent antibacterial activity against Staphylococcus aureus or Escherichia coli, which could reduce the incidence of skin wound infections. Collectively, we demonstrate that sprayable PF127 could be used as a new drug delivery system for treatment of skin injury. The advantages of this sprayable system are obvious: (1) It is convenient to use; (2) The hydrogel can cover irregular skin defect sites evenly in a liquid state. In combination with this system, we establish a novel sprayable adhesive (ZnMet-PF127) and demonstrate that it is a potential clinical treatment for traumatic skin defect and burn skin injury.

Mesenchymal stem cells in fibrotic diseases-the two sides of the same coin.

Fibrosis is caused by extensive deposition of extracellular matrix (ECM) components, which play a crucial role in injury repair. Fibrosis attributes to ~45% of all deaths worldwide. The molecular pathology of different fibrotic diseases varies, and a number of bioactive factors are involved in the pathogenic process. Mesenchymal stem cells (MSCs) are a type of multipotent stem cells that have promising therapeutic effects in the treatment of different diseases. Current updates of fibrotic pathogenesis reveal that residential MSCs may differentiate into myofibroblasts which lead to the fibrosis development. However, preclinical and clinical trials with autologous or allogeneic MSCs infusion demonstrate that MSCs can relieve the fibrotic diseases by modulating inflammation, regenerating damaged tissues, remodeling the ECMs, and modulating the death of stressed cells after implantation. A variety of animal models were developed to study the mechanisms behind different fibrotic tissues and test the preclinical efficacy of MSC therapy in these diseases. Furthermore, MSCs have been used for treating liver cirrhosis and pulmonary fibrosis patients in several clinical trials, leading to satisfactory clinical efficacy without severe adverse events. This review discusses the two opposite roles of residential MSCs and external MSCs in fibrotic diseases, and summarizes the current perspective of therapeutic mechanism of MSCs in fibrosis, through both laboratory study and clinical trials.

Structure-guided optimization of D-captopril for discovery of potent NDM-1 inhibitors.

ß-lactam antibiotics have long been the mainstay for the treatment of bacterial infections. New Delhi metallo-ß-lactamase 1 (NDM-1) is able to hydrolyze nearly all ß-lactam antibiotics and even clinically used serine-ß-lactamase inhibitors. The wide and rapid spreading of NDM-1 gene among pathogenic bacteria has attracted extensive attention, therefore high potency NDM-1 inhibitors are urgently needed. Here we report a series of structure-guided design of D-captopril derivatives that can inhibit the activity of NDM-1 in vitro and at cellular levels. Structural comparison indicates the mechanisms of inhibition enhancement and provides insights for further inhibitor optimization.

Functional Identification and Structural Analysis of a New Lipoate Protein Ligase in Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the causative agent of pandemic pneumonia among pigs, namely, swine enzootic pneumonia. Although M. hyopneumoniae was first identified in 1965, little is known regarding its metabolic pathways, which might play a pivotal role during disease pathogenesis. Lipoate is an essential cofactor for enzymes important for central metabolism. However, the lipoate metabolism pathway in M. hyopneumoniae is definitely unclear. Here, we identified a novel gene, lpl, encoding a lipoate protein ligase in the genome of M. hyopneumoniae (Mhp-Lpl). This gene contains 1,032 base pairs and encodes a protein of 343 amino acids, which is between 7.5 and 36.09% identical to lipoate protein ligases (Lpls) of other species. Similar to its homologs in other species, Mhp-Lpl catalyzes the ATP-dependent activation of lipoate to lipoyl-AMP and the transfer of the activated lipoyl onto the lipoyl domains of M. hyopneumoniae GcvH (Mhp H) in vitro. Enzymatic and mutagenesis analysis indicate that residue K56 within the SKT sequence of Mhp H protein is the lipoyl moiety acceptor site. The three-dimensional structure showed typical lipoate protein ligase folding, with a large N-terminal domain and a small C-terminal domain. The large N-terminal domain is responsible for the full enzymatic activity of Mhp-Lpl. The identification and characterization of Mhp-Lpl will be beneficial to our understanding of M. hyopneumoniae metabolism. Summary: Lipoic acid is an essential cofactor for the activation of some enzyme complexes involved in key metabolic processes. Lipoate protein ligases (Lpls) are responsible for the metabolism of lipoic acid. To date, little is known regarding the Lpls in M. hyopneumoniae. In this study, we identified a lipoate protein ligase of M. hyopneumoniae. We further analyzed the function, overall structure and ligand-binding site of this protein. The lipoate acceptor site on M. hyopneumoniae GcvH was also identified. Together, these findings reveal that Lpl exists in M. hyopneumoniae and will provide a basis for further exploration of the pathway of lipoic acid metabolism in M. hyopneumoniae.

N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides bearing heteroaromatic rings as novel antibacterial agents: Design, synthesis, biological evaluation and target identification.

Due to the occurrence of antibiotic resistance, bacterial infectious diseases have become a serious threat to public health. To overcome antibiotic resistance, novel antibiotics are urgently needed. N-thiadiazole-4-hydroxy-2-quinolone-3-carboxamides are a potential new class of antibacterial agents, as one of its derivatives was identified as an antibacterial agent against S. aureus. However, no potency-directed structural optimization has been performed. In this study, we designed and synthesized 37 derivatives, and evaluated their antibacterial activity against S. aureus ATCC29213, which led to the identification of ten potent antibacterial agents with minimum inhibitory concentration (MIC) values below 1 µg/mL. Next, we performed bacterial growth inhibition assays against a panel of drug-resistant clinical isolates, including methicillin-resistant S. aureus, and cytotoxicity assays with HepG2 and HUVEC cells. One of the tested compounds named 1-ethyl-4-hydroxy-2-oxo-N-(5-(thiazol-2-yl)-1,3,4-thiadiazol-2-yl)-1,2-dihydroquinoline-3-carboxamide (g37) showed 2 to 128-times improvement compared with vancomycin in term of antibacterial potency against the tested strains (MICs: 0.25-1 µg/mL vs. 1-64 µg/mL) and an optimal selective toxicity (HepG2/MRSA, 110.6 to 221.2; HUVEC/MRSA, 77.6-155.2). Further, comprehensive evaluation indicated that g37 did not induce resistance development of MRSA over 20 passages, and it has been confirmed as a bactericidal, metabolically stable, orally active antibacterial agent. More importantly, we have identified the S. aureus DNA gyrase B as its potential target and proposed a potential binding mode by molecular docking. Taken together, the present work reports the most potent derivative of this chemical series (g37) and uncovers its potential target, which lays a solid foundation for further lead optimization facilitated by the structure-based drug design technique.

Bacterial Lipoprotein Biosynthetic Pathway as a Potential Target for Structure-based Design of Antibacterial Agents.

BACKGROUND: Antibiotic resistance is currently a serious problem for global public health. To this end, discovery of new antibacterial drugs that interact with novel targets is important. The biosynthesis of lipoproteins is vital to bacterial survival and its inhibitors have shown efficacy against a range of bacteria, thus bacterial lipoprotein biosynthetic pathway is a potential target. METHODS: At first, the literature that covered the basic concept of bacterial lipoprotein biosynthetic pathway as well as biochemical characterization of three key enzymes was reviewed. Then, the recently resolved crystal structures of the three enzymes were retrieved from Protein Data Bank (PDB) and the essential residues in the active sites were analyzed. Lastly, all the available specific inhibitors targeting this pathway and their Structure-activity Relationship (SAR) were discussed. RESULTS: We briefly introduce the bacterial lipoprotein biosynthetic pathway and describe the structures and functions of three key enzymes in detail. In addition, we present much knowledge on ligand recognition that may facilitate structure-based drug design. Moreover, we focus on the SAR of LspA inhibitors and discuss their potency and drug-likeness. CONCLUSION: This review presents a clear background of lipoprotein biosynthetic pathway and provides practical clues for structure-based drug design. In particular, the most up-to-date knowledge on the SAR of lead compounds targeting this pathway would be a good reference for discovery of a novel class of antibacterial agents.

Active-Site Conformational Fluctuations Promote the Enzymatic Activity of NDM-1.

ß-Lactam antibiotics are the mainstay for the treatment of bacterial infections. However, elevated resistance to these antibiotics mediated by metallo-ß-lactamases (MBLs) has become a global concern. New Delhi metallo-ß-lactamase-1 (NDM-1), a newly added member of the MBL family that can hydrolyze almost all ß-lactam antibiotics, has rapidly spread all over the world and poses serious clinical threats. Broad-spectrum and mechanism-based inhibitors against all MBLs are highly desired, but the differential mechanisms of MBLs toward different antibiotics pose a great challenge. To facilitate the design of mechanism-based inhibitors, we investigated the active-site conformational changes of NDM-1 through the determination of a series of 15 high-resolution crystal structures in native form and in complex with products and by using biochemical and biophysical studies, site-directed mutagenesis, and molecular dynamics computation. The structural studies reveal the consistency of the active-site conformations in NDM-1/product complexes and the fluctuation in native NDM-1 structures. The enzymatic measurements indicate a correlation between enzymatic activity and the active-site fluctuation, with more fluctuation favoring higher activity. This correlation is further validated by structural and enzymatic studies of the Q123G mutant. Our combinational studies suggest that active-site conformational fluctuation promotes the enzymatic activity of NDM-1, which may guide further mechanism studies and inhibitor design.

A dual-specific IGF-I/II human engineered antibody domain inhibits IGF signaling in breast cancer cells.

The insulin-like growth factors (IGFs), IGF-I and IGF-II, are essential for regulating cell growth, differentiation and metastasis of a broad range of malignancies. The IGF-I/II actions are mediated through the IGF receptor type 1 (IGF-1R) and the insulin receptor (IR), which are overexpressed in multiple types of tumors. Here, we have firstly identified a human engineered antibody domain (eAd) from a phage-displayed VH library. The eAd suppressed the signal transduction of IGF-1R mediated by exogenous IGF-I or IGF-II in breast cancer cell lines through neutralizing both IGF-I and IGF-II. It also significantly inhibited the growth of breast cancer cells. Therefore, the anti-IGF-I/II eAd offers an alternative approach to target both the IGF-1R signaling pathways through the inhibition of IGF-I/II.

Bismuth antimicrobial drugs serve as broad-spectrum metallo-ß-lactamase inhibitors.

Drug-resistant superbugs pose a huge threat to human health. Infections by Enterobacteriaceae producing metallo-ß-lactamases (MBLs), e.g., New Delhi metallo-ß-lactamase 1 (NDM-1) are very difficult to treat. Development of effective MBL inhibitors to revive the efficacy of existing antibiotics is highly desirable. However, such inhibitors are not clinically available till now. Here we show that an anti-Helicobacter pylori drug, colloidal bismuth subcitrate (CBS), and related Bi(III) compounds irreversibly inhibit different types of MBLs via the mechanism, with one Bi(III) displacing two Zn(II) ions as revealed by X-ray crystallography, leading to the release of Zn(II) cofactors. CBS restores meropenem (MER) efficacy against MBL-positive bacteria in vitro, and in mice infection model, importantly, also slows down the development of higher-level resistance in NDM-1-positive bacteria. This study demonstrates a high potential of Bi(III) compounds as the first broad-spectrum B1 MBL inhibitors to treat MBL-positive bacterial infection in conjunction with existing carbapenems.

High resolution crystal structure of the catalytic domain of MCR-1.

The newly identified mobile colistin resistant gene (mcr-1) rapidly spread among different bacterial strains and confers colistin resistance to its host, which has become a global concern. Based on sequence alignment, MCR-1 should be a phosphoethanolamine transferase, members of the YhjW/YjdB/YijP superfamily and catalyze the addition of phosphoethanolamine to lipid A, which needs to be validated experimentally. Here we report the first high-resolution crystal structure of the C-terminal catalytic domain of MCR-1 (MCR-1C) in its native state. The active pocket of native MCR-1C depicts unphosphorylated nucleophilic residue Thr285 in coordination with two Zinc ions and water molecules. A flexible adjacent active site loop (aa: Lys348-365) pose an open conformation compared to its structural homologues, suggesting of an open substrate entry channel. Taken together, this structure sets ground for further study of substrate binding and MCR-1 catalytic mechanism in development of potential therapeutic agents.