A bimolecular modification strategy for developing long-lasting bone anabolic aptamer.

The molecular weight of nucleic acid aptamers (20 kDa) is lower than the cutoff threshold of the renal filtration (30-50 kDa), resulting in a very short half-life, which dramatically limits their druggability. To address this, we utilized 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-hydroxy-2-oxo-2H-chromen-6-yl)propenamide (HC) and 12-((2,5-dioxopyrrolidin-1-yl)oxy)-12-oxododecanoic acid (DA), two newly designed coupling agents, for synergistic binding to human serum albumin (HSA). Both HC and DA are conjugated to a bone anabolic aptamer (Apc001) against sclerostin to form an Apc001OC conjugate with high binding affinity to HSA. Notably, HC and DA could synergistically facilitate prolonging the half-life of the conjugated Apc001 and promoting its bone anabolic potential. Using the designed blocking peptides, the mechanism studies indicate that the synergistic effect of HC-DA on pharmacokinetics and bone anabolic potential of the conjugated Apc001 is achieved via their synergistic binding to HSA. Moreover, biweekly Apc001OC at 50 mg/kg shows comparable bone anabolic potential to the marketed sclerostin antibody given weekly at 25 mg/kg. This proposed bimolecular modification strategy could help address the druggability challenge for aptamers with a short half-life.

The role of sclerostin in lipid and glucose metabolism disorders.

Lipid and glucose metabolism are critical for human activities, and their disorders can cause diabetes and obesity, two prevalent metabolic diseases. Studies suggest that the bone involved in lipid and glucose metabolism is emerging as an endocrine organ that regulates systemic metabolism through bone-derived molecules. Sclerostin, a protein mainly produced by osteocytes, has been therapeutically targeted by antibodies for treating osteoporosis owing to its ability to inhibit bone formation. Moreover, recent evidence indicates that sclerostin plays a role in lipid and glucose metabolism disorders. Although the effects of sclerostin on bone have been extensively examined and reviewed, its effects on systemic metabolism have not yet been well summarized. In this paper, we provide a systemic review of the effects of sclerostin on lipid and glucose metabolism based on in vitro and in vivo evidence, summarize the research progress on sclerostin, and prospect its potential manipulation for obesity and diabetes treatment.

RNA-targeted small-molecule drug discoveries: a machine-learning perspective.

In the past two decades, machine learning (ML) has been extensively adopted in protein-targeted small molecule (SM) discovery. Once trained, ML models could exert their predicting abilities on large volumes of molecules within a short time. However, applying ML approaches to discover RNA-targeted SMs is still in its early stages. This is primarily because of the intrinsic structural instability of RNA molecules that impede the structure-based screening or designing of RNA-targeted SMs. Recently, with more studies revealing RNA structures and a growing number of RNA-targeted ligands being identified, it resulted in an increased interest in the field of drugging RNA. Undeniably, intracellular RNA is much more abundant than protein and, if successfully targeted, will be a major alternative target for therapeutics. Therefore, in this context, as well as under the premise of having RNA-related research data, ML-based methods can get involved in improving the speed of traditional experimental processes. [Figure: see text].

Therapeutic aptamer targeting sclerostin loop3 for promoting bone formation without increasing cardiovascular risk in osteogenesis imperfecta mice.

Rationale: Sclerostin inhibition demonstrated bone anabolic potential in osteogenesis imperfecta (OI) mice, whereas humanized therapeutic sclerostin antibody romosozumab for postmenopausal osteoporosis imposed clinically severe cardiac ischemic events. Therefore, it is desirable to develop the next generation sclerostin inhibitors to promote bone formation without increasing cardiovascular risk for OI. Methods and Results: Our data showed that sclerostin suppressed inflammatory responses, prevented aortic aneurysm (AA) and atherosclerosis progression in hSOSTki.Col1a2+/G610C.ApoE-/- mice. Either loop2&3 deficiency or inhibition attenuated sclerostin's suppressive effects on expression of inflammatory cytokines and chemokines in vitro, whilst loop3 deficiency maintained the protective effect of sclerostin on cardiovascular system both in vitro and in vivo. Moreover, loop3 was critical for sclerostin's antagonistic effect on bone formation in Col1a2+/G610C mice. Accordingly, a sclerostin loop3-specific aptamer aptscl56 was identified by our lab. It could recognize both recombinant sclerostin and sclerostin in the serum of OI patients via targeting loop3. PEG40k conjugated aptscl56 (Apc001PE) demonstrated to promote bone formation, increase bone mass and improve bone microarchitecture integrity in Col1a2+/G610C mice via targeting loop3, while did not show influence in inflammatory response, AA and atherosclerosis progression in Col1a2+/G610C.ApoE-/- mice with Angiotensin II infusion. Further, Apc001PE had no influence in the protective effect of sclerostin on cardiovascular system in hSOSTki.Col1a2+/G610C.ApoE-/- mice, while it inhibited the antagonistic effect of sclerostin on bone formation in hSOSTki.Col1a2+/G610C mice via targeting loop3. Apc001PE was non-toxic to healthy rodents, even at ultrahigh dose. Apc001PE for OI was granted orphan drug designation by US-FDA in 2019 (DRU-2019-6966). Conclusion: Sclerostin loop3-specific aptamer Apc001PE promoted bone formation without increasing cardiovascular risk in OI mice.

Targeting loop3 of sclerostin preserves its cardiovascular protective action and promotes bone formation.

Sclerostin negatively regulates bone formation by antagonizing Wnt signalling. An antibody targeting sclerostin for the treatment of postmenopausal osteoporosis was approved by the U.S. Food and Drug Administration, with a boxed warning for cardiovascular risk. Here we demonstrate that sclerostin participates in protecting cardiovascular system and inhibiting bone formation via different loops. Loop3 deficiency by genetic truncation could maintain sclerostin's protective effect on the cardiovascular system while attenuating its inhibitory effect on bone formation. We identify an aptamer, named aptscl56, which specifically targets sclerostin loop3 and use a modified aptscl56 version, called Apc001PE, as specific in vivo pharmacologic tool to validate the above effect of loop3. Apc001PE has no effect on aortic aneurysm and atherosclerotic development in ApoE-/- mice and hSOSTki.ApoE-/- mice with angiotensin II infusion. Apc001PE can promote bone formation in hSOSTki mice and ovariectomy-induced osteoporotic rats. In summary, sclerostin loop3 cannot participate in protecting the cardiovascular system, but participates in inhibiting bone formation.

Drug discovery of sclerostin inhibitors.

Sclerostin, a protein secreted from osteocytes, negatively regulates the WNT signaling pathway by binding to the LRP5/6 co-receptors and further inhibits bone formation and promotes bone resorption. Sclerostin contributes to musculoskeletal system-related diseases, making it a promising therapeutic target for the treatment of WNT-related bone diseases. Additionally, emerging evidence indicates that sclerostin contributes to the development of cancers, obesity, and diabetes, suggesting that it may be a promising therapeutic target for these diseases. Notably, cardiovascular diseases are related to the protective role of sclerostin. In this review, we summarize three distinct types of inhibitors targeting sclerostin, monoclonal antibodies, aptamers, and small-molecule inhibitors, from which monoclonal antibodies have been developed. As the first-in-class sclerostin inhibitor approved by the U.S. FDA, the monoclonal antibody romosozumab has demonstrated excellent effectiveness in the treatment of postmenopausal osteoporosis; however, it conferred high cardiovascular risk in clinical trials. Furthermore, romosozumab could only be administered by injection, which may cause compliance issues for patients who prefer oral therapy. Considering these above safety and compliance concerns, we therefore present relevant discussion and offer perspectives on the development of next-generation sclerostin inhibitors by following several ways, such as concomitant medication, artificial intelligence-based strategy, druggable modification, and bispecific inhibitors strategy.

A Rapid Protocol for Direct Isolation of Osteoclast Lineage Cells from Mouse Bone Marrow.

Osteoclast lineage cells (OLCs), including osteoclast precursors (OCPs) and mature osteoclasts (MOCs), participate in bone remodeling and mediate pathologic bone loss. Thus, it is essential to obtain OLCs for exploring their molecular features in both physiological and pathological conditions in vivo. However, the conventional protocols for obtaining OLCs ex vivo are not only time-consuming, but also unable to capture the cellular status of OLCs in vivo. In addition, the current antibody-based isolation approaches, such as fluorescence-/ magnetic-activated cell sorting, are not able to obtain pure osteoclasts because no unique surface antigen for osteoclasts has been identified. Here, we develop a rapid protocol for directly isolating OLCs from mouse bone marrow through magnetic-activated cell sorting (MACS). This protocol can rapidly enrich OCPs and MOCs, respectively, depending on the expression of the distinctive surface markers at their differentiation stages. It is optimized to isolate OLCs from four mice concurrently, of which sorting procedure could be completed within ~5 h.

Drug Discovery of DKK1 Inhibitors.

Dickkopf-1 (DKK1) is a well-characterized Wnt inhibitor and component of the Wnt/ß-catenin signaling pathway, whose dysregulation is associated with multiple abnormal pathologies including osteoporosis, Alzheimer's disease, diabetes, and various cancers. The Wnt signaling pathway has fundamental roles in cell fate determination, cell proliferation, and survival; thus, its mis-regulation can lead to disease. Although DKK1 is involved in other signaling pathways, including the ß-catenin-independent Wnt pathway and the DKK1/CKAP4 pathway, the inhibition of DKK1 to propagate Wnt/ß-catenin signals has been validated as an effective way to treat related diseases. In fact, strategies for developing DKK1 inhibitors have produced encouraging clinical results in different pathological models, and many publications provide detailed information about these inhibitors, which include small molecules, antibodies, and nucleic acids, and may function at the protein or mRNA level. However, no systematic review has yet provided an overview of the various aspects of their development and prospects. Therefore, we review the DKK1 inhibitors currently available or under study and provide an outlook on future studies involving DKK1 and drug discovery.

Current Pharmacological Strategies for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder caused by the absence of dystrophin protein, which is essential for muscle fiber integrity. Loss of dystrophin protein leads to recurrent myofiber damage, chronic inflammation, progressive fibrosis, and dysfunction of muscle stem cells. There is still no cure for DMD so far and the standard of care is principally limited to symptom relief through glucocorticoids treatments. Current therapeutic strategies could be divided into two lines. Dystrophin-targeted therapeutic strategies that aim at restoring the expression and/or function of dystrophin, including gene-based, cell-based and protein replacement therapies. The other line of therapeutic strategies aims to improve muscle function and quality by targeting the downstream pathological changes, including inflammation, fibrosis, and muscle atrophy. This review introduces the important developments in these two lines of strategies, especially those that have entered the clinical phase and/or have great potential for clinical translation. The rationale and efficacy of each agent in pre-clinical or clinical studies are presented. Furthermore, a meta-analysis of gene profiling in DMD patients has been performed to understand the molecular mechanisms of DMD.

Dickkopf-1: A Promising Target for Cancer Immunotherapy.

Clinical studies in a range of cancers have detected elevated levels of the Wnt antagonist Dickkopf-1 (DKK1) in the serum or tumors of patients, and this was frequently associated with a poor prognosis. Our analysis of DKK1 gene profile using data from TCGA also proves the high expression of DKK1 in 14 types of cancers. Numerous preclinical studies have demonstrated the cancer-promoting effects of DKK1 in both in vitro cell models and in vivo animal models. Furthermore, DKK1 showed the ability to modulate immune cell activities as well as the immunosuppressive cancer microenvironment. Expression level of DKK1 is positively correlated with infiltrating levels of myeloid-derived suppressor cells (MDSCs) in 20 types of cancers, while negatively associated with CD8+ T cells in 4 of these 20 cancer types. Emerging experimental evidence indicates that DKK1 has been involved in T cell differentiation and induction of cancer evasion of immune surveillance by accumulating MDSCs. Consequently, DKK1 has become a promising target for cancer immunotherapy, and the mechanisms of DKK1 affecting cancers and immune cells have received great attention. This review introduces the rapidly growing body of literature revealing the cancer-promoting and immune regulatory activities of DKK1. In addition, this review also predicts that by understanding the interaction between different domains of DKK1 through computational modeling and functional studies, the underlying functional mechanism of DKK1 could be further elucidated, thus facilitating the development of anti-DKK1 drugs with more promising efficacy in cancer immunotherapy.

Structural Biology for the Molecular Insight between Aptamers and Target Proteins.

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer-target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer-protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.

Exosomal transfer of osteoclast-derived miRNAs to chondrocytes contributes to osteoarthritis progression.

Osteoarthritis (OA) is a prevalent aging-related joint disease lacking disease-modifying therapies. Here, we identified an upregulation of circulating exosomal osteoclast (OC)-derived microRNAs (OC-miRNAs) during the progression of surgery-induced OA in mice. We found that reducing OC-miRNAs by Cre-mediated excision of the key miRNA-processing enzyme Dicer or blocking the secretion of OC-originated exosomes by short interfering RNA-mediated silencing of Rab27a substantially delayed the progression of surgery-induced OA in mice. Mechanistically, the exosomal transfer of OC-miRNAs to chondrocytes reduced the resistance of cartilage to matrix degeneration, osteochondral angiogenesis and sensory innervation during OA progression by suppressing tissue inhibitor of metalloproteinase-2 (TIMP-2) and TIMP-3. Furthermore, systemic administration of a new OC-targeted exosome inhibitor (OCExoInhib) blunted the progression of surgery-induced OA in mice. We suggest that targeting the exosomal transfer of OC-miRNAs to chondrocytes represents a potential therapeutic avenue to tackle OA progression.

Connective Tissue Growth Factor: From Molecular Understandings to Drug Discovery.

Connective tissue growth factor (CTGF) is a key signaling and regulatory molecule involved in different biological processes, such as cell proliferation, angiogenesis, and wound healing, as well as multiple pathologies, such as tumor development and tissue fibrosis. Although the underlying mechanisms of CTGF remain incompletely understood, a commonly accepted theory is that the interactions between different protein domains in CTGF and other various regulatory proteins and ligands contribute to its variety of functions. Here, we highlight the structure of each domain of CTGF and its biology functions in physiological conditions. We further summarized main diseases that are deeply influenced by CTGF domains and the potential targets of these diseases. Finally, we address the advantages and disadvantages of current drugs targeting CTGF and provide the perspective for the drug discovery of the next generation of CTGF inhibitors based on aptamers.

A Loop-Based and AGO-Incorporated Virtual Screening Model Targeting AGO-Mediated miRNA-mRNA Interactions for Drug Discovery to Rescue Bone Phenotype in Genetically Modified Mice.

Several virtual screening models are proposed to screen small molecules only targeting primary miRNAs without selectivity. Few attempts have been made to develop virtual screening strategies for discovering small molecules targeting mature miRNAs. Mature miRNAs and their specific target mRNA can form unique functional loops during argonaute (AGO)-mediated miRNA-mRNA interactions, which may serve as potential targets for small-molecule drug discovery. Thus, a loop-based and AGO-incorporated virtual screening model is constructed for targeting the loops. The previously published studies have found that miR-214 can target ATF4 to inhibit osteoblastic bone formation, whereas miR-214 can target TRAF3 to promote osteoclast activity. By using the virtual model, the top ten candidate small molecules targeting miR-214-ATF4 mRNA interactions and top ten candidate small molecules targeting miR-214-TRAF3 mRNA interactions are selected, respectively. Based on both in vitro and in vivo data, one small molecule can target miR-214-ATF4 mRNA to promote ATF4 protein expression and enhance osteogenic potential, whereas one small molecule can target miR-214-TRAF3 mRNA to promote TRAF3 protein expression and inhibit osteoclast activity. These data indicate that the loop-based and AGO-incorporated virtual screening model can help to obtain small molecules specifically targeting miRNA-mRNA interactions to rescue bone phenotype in genetically modified mice.

Pros and Cons of Denosumab Treatment for Osteoporosis and Implication for RANKL Aptamer Therapy.

Osteoporosis is age-related deterioration in bone mass and micro-architecture. Denosumab is a novel human monoclonal antibody for osteoporosis. It is a receptor activator of nuclear factor-κB ligand (RANKL) inhibitor, which binds to and inhibits osteoblast-produced RANKL, in turn reduces the binding between RANKL and osteoclast receptor RANK, therefore decreases osteoclast-mediated bone resorption and turnover. However, adverse events have also been reported after denosumab treatment, including skin eczema, flatulence, cellulitis and osteonecrosis of the jaw (ONJ). Extensive researches on the mechanism of adverse reactions caused by denosumab have been conducted and may provide new insights into developing new RANKL inhibitors that achieve better specificity and safety. Aptamers are single-stranded oligonucleotides that can bind to target molecules with high specificity and affinity. They are screened from large single-stranded synthetic oligonucleotides and enriched by a technology named SELEX (systematic evolution of ligands by exponential enrichment). With extra advantages such as high stability, low immunogenicity and easy production over antibodies, aptamers are hypothesized to be promising candidates for therapeutic drugs targeting RANKL to counteract osteoporosis. In this review, we focus on the pros and cons of denosumab treatment in osteoporosis and the implication for novel aptamer treatment.

YY1 regulates skeletal muscle regeneration through controlling metabolic reprogramming of satellite cells.

Skeletal muscle satellite cells (SCs) are adult muscle stem cells responsible for muscle regeneration after acute or chronic injuries. The lineage progression of quiescent SC toward activation, proliferation, and differentiation during the regeneration is orchestrated by cascades of transcription factors (TFs). Here, we elucidate the function of TF Yin Yang1 (YY1) in muscle regeneration. Muscle-specific deletion of YY1 in embryonic muscle progenitors leads to severe deformity of diaphragm muscle formation, thus neonatal death. Inducible deletion of YY1 in SC almost completely blocks the acute damage-induced muscle repair and exacerbates the chronic injury-induced dystrophic phenotype. Examination of SC revealed that YY1 loss results in cell-autonomous defect in activation and proliferation. Mechanistic search revealed that YY1 binds and represses mitochondrial gene expression. Simultaneously, it also stabilizes Hif1α protein and activates Hif1α-mediated glycolytic genes to facilitate a metabolic reprogramming toward glycolysis which is needed for SC proliferation. Altogether, our findings have identified YY1 as a key regulator of SC metabolic reprogramming through its dual roles in modulating both mitochondrial and glycolytic pathways.

Bushen Yijing Fang Reduces Fall Risk in Late Postmenopausal Women with Osteopenia: A Randomized Double-blind and Placebo-controlled Trial.

Falls in late postmenopausal women with osteopenia usually cause fractures with severe consequences. This 36-month randomized, double-blind and placebo-controlled trial with a 10-year observational follow-up study aimed to investigate the long-term effect of herbal formula Bushen Yijing Fang (BSYJF) on fall risk in the late postmenopausal women with osteopenia. 140 late postmenopausal women (Femoral neck T-score, -2.5~-2 SD) were recruited and randomized to orally receive calcium carbonate 300 mg daily with either BSYJF or placebo for 36 months. The effect was further investigated for another 10-year follow-up. During the 36-month administration, there were 12 falls in BSYJF group and 28 falls in placebo group, respectively, indicating 64% lower risk of falls (RR 0.36 [95% CI, 0.18 to 0.71]; P = 0.004) in BSYJF group. During the 10-year follow-up, 36% lower fall risk (RR 0.64 [95% CI, 0.46 to 0.89]; P = 0.009) was observed in BSYJF group. No significant difference was found in safety profile between two groups. Thirty-six-month administration of BSYJF reduced fall risk with an increase in bone mass, and its latent effect on fall risk was continually observed in the 10-year follow-up in late postmenopausal women with osteopenia. This clinical trial was registered at Chinese clinical trial registry (ChiCTR-IOR-16008942).

Long Noncoding RNA lncMUMA Reverses Established Skeletal Muscle Atrophy following Mechanical Unloading.

Reversing established muscle atrophy following mechanical unloading is of great clinical challenge. Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in myogenesis. Here we identified a lncRNA (mechanical unloading-induced muscle atrophy-related lncRNA [lncMUMA]) enriched in muscle, which was the most downregulated lncRNA during muscle atrophy development in hindlimb suspension (HLS) mice. The in vitro and in vivo data demonstrated that the decreased expression levels of lncMUMA closely associated with a reduction of myogenesis during mechanical unloading. Mechanistically, lncMUMA promoted myogenic differentiation by functioning as a miR-762 sponge to regulate the core myogenic regulator MyoD in vitro. The enforced expression of lncMUMA relieved the decreases in MyoD protein and muscle mass in miR-762 knockin mice. Therapeutically, the enforced expression of lncMUMA improved the in vitro myogenic differentiation of myoblasts under microgravity simulation, prevented the muscle atrophy development, and reversed the established muscle atrophy in HLS mice. These findings identify lncMUMA as an anabolic regulator to reverse established muscle atrophy following mechanical unloading.

A newly identified lncRNA MAR1 acts as a miR-487b sponge to promote skeletal muscle differentiation and regeneration.

BACKGROUND: Skeletal muscle atrophy induced by either aging (sarcopenia) or mechanical unloading is associated with serious health consequences. Long non-coding RNAs (lncRNAs) are implicated as important regulators in numerous physiological and pathological processes. METHODS: Microarray analysis was performed to identify the differentially expressed lncRNAs in skeletal muscle between adult and aged mice. The most decreased lncRNA in aged skeletal muscle was identified. The C2C12 mouse myoblast cells were used to assess the biological function of the lncRNA in vitro. The target microRNA of lncRNA and the target protein of microRNA were predicted by bioinformatics analysis and validated in vitro. Furthermore, the biology function of the lncRNA in vivo was investigated by local overexpression or knockdown the lncRNA in skeletal muscle. The therapeutic effect of the lncRNA overexpression in age-related or mechanical unloading-induced muscle atrophy was also evaluated. RESULTS: We identified a novel lncRNA (muscle anabolic regulator 1, MAR1) which was highly expressed in mice skeletal muscle and positively correlated with muscle differentiation and growth in vitro and in vivo. We predicted and validated that microRNA-487b (miR-487b) was a direct target of MAR1. We also predicted and validated that Wnt5a, an important regulator during myogenesis, was a target of miR-487b in C2C12 cells. Our findings further demonstrated that enforced MAR1 expression in myoblasts led to derepression of Wnt5a. Moreover, MAR1 promoted skeletal muscle mass/strength and Wnt5a protein level in mice. Enforced MAR1 expression in mice attenuated muscle atrophy induced by either aging or unloading. CONCLUSIONS: The newly identified lncRNA MAR1 acts as a miR-487b sponge to regulate Wnt5a protein, resulting in promoting muscle differentiation and regeneration. MAR1 could be a novel therapeutic target for treating muscle atrophy induced by either aging or mechanical unloading.

A water-soluble nucleolin aptamer-paclitaxel conjugate for tumor-specific targeting in ovarian cancer.

Paclitaxel (PTX) is among the most commonly used first-line drugs for cancer chemotherapy. However, its poor water solubility and indiscriminate distribution in normal tissues remain clinical challenges. Here we design and synthesize a highly water-soluble nucleolin aptamer-paclitaxel conjugate (NucA-PTX) that selectively delivers PTX to the tumor site. By connecting a tumor-targeting nucleolin aptamer (NucA) to the active hydroxyl group at 2' position of PTX via a cathepsin B sensitive dipeptide bond, NucA-PTX remains stable and inactive in the circulation. NucA facilitates the uptake of the conjugated PTX specifically in tumor cells. Once inside cells, the dipeptide bond linker of NucA-PTX is cleaved by cathepsin B and then the conjugated PTX is released for action. The NucA modification assists the selective accumulation of the conjugated PTX in ovarian tumor tissue rather than normal tissues, and subsequently resulting in notably improved antitumor activity and reduced toxicity.