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As persistent elevation of transforming growth factor‐β (TGF‐β) promotes fibrosis of muscles and joints and accelerates disease progression in amyotrophic lateral sclerosis (ALS), we investigated whether inhibition of TGF‐β would be effective against both exacerbations. The effects of TGF‐β and its inhibitor on myoblasts and fibroblasts were tested in vitro and confirmed in vivo, and the dual action of a TGF‐β inhibitor in ameliorating the pathogenic role of TGF‐β in ALS mice was identified. In the peripheral neuromuscular system, fibrosis in the muscles and joint cavities induced by excessive TGF‐β causes joint contracture and muscular degeneration, which leads to motor dysfunction. In an ALS mouse model, an increase in TGF‐β in the central nervous system (CNS), consistent with astrocyte activity, was associated with M1 microglial activity and pro‐inflammatory conditions, as well as with neuronal cell death. Treatment with the TGF‐β inhibitor halofuginone could prevent musculoskeletal fibrosis, resulting in the alleviation of joint contracture and delay of motor deterioration in ALS mice. Halofuginone could also reduce glial cell‐induced neuroinflammation and neuronal apoptosis. These dual therapeutic effects on both the neuromuscular system and the CNS were observed from the beginning to the end stages of ALS; as a result, treatment with a TGF‐β inhibitor from the early stage of disease delayed the time of symptom exacerbation in ALS mice, which led to prolonged survival. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. Halofuginone (HF) exerts beneficial effects on organ fibrosis, periodontitis, and cancer. However, the effect of HF against AF remains unknown. During the induction of AF, the rats were intragastrically administered HF (5 mg/kg and 10 mg/kg) daily for 7 consecutive days. Cardiac function was evaluated through echocardiographic analysis. The presence of pathological changes and interstitial fibrosis in the left atrial tissues were investigated. Intracellular Ca2+ homeostasis and mitochondrial function in atrial tissues were evaluated. The activation of the PI3K/Akt signaling pathway was examined, and an allosteric Akt inhibitor, MK-2206, was applied to confirm the involvement of the PI3K/Akt signaling pathway in the protection against AF by HF. The administration of HF resulted in a prolongation of the atrial effective refractory period (AERP), a reduction in both the duration and inducibility of AF, and a decrease in atrial weight, heart weight, atrial weight/body weight ratio, and heart weight/body weight ratio in rats with AF. In addition, the administration of HF resulted in a reduction in left atrial diameter (LAD) and an increase in left ventricular internal diameter diastolic (LVIDd), ejection fraction (EF), and fractional shortening (FS), while having no effect on left ventricular internal diameter systolic (LVIDs). The pathological changes and cardiac fibrosis observed in rats with AF were mitigated by HF. Moreover, HF enhanced mitochondrial function, suppressed cardiomyocyte apoptosis, and activated the PI3K/Akt pathway in AF rats. Furthermore, the protective effect against AF was also observed in an in vitro model. The effects of HF on fibrosis markers, intracellular Ca2+ homeostasis, mitochondrial function, and cardiac apoptosis were blocked by MK-2206. HF alleviated the susceptibility to AF in vivo and in vitro via the activation of the PI3K/Akt signaling pathway. [ABSTRACT FROM AUTHOR]

Evidences shows that T helper 17 (Th17) and regulatory T (Treg) cells imbalance plays a critical role in bone lesions of MM patients. Therefore, regulating the Th17/Treg imbalance may be beneficial for bone lesions in MM. Ten MM mice complicated with bone lesions were established and divided into the halofuginone (HF) group and the PBS group. After treatment, tibia and fibula from both groups were scanned by micro-CT. Osteoclasts and osteoblasts were validated by histochemical staining and ELISA. Th17 and Treg cells were tested by flow cytometry. The correlations between Th17/Treg cell ratio and osteoclasts, osteoblasts and bone remodeling were analyzed using the Spearman relative analysis. After treatment, mice in the HF group had an increase in trabecular bone volume fraction and thickened cortex, but a decrease in trabecular separation compared to mice in the PBS group.Tartrate-resistant acid phosphase (TRAP) + osteoclasts and its biomarker TRACP5b in serum were reduced, while alkaline phosphatase (ALP) + osteoblasts and its biomarker N-terminal propeptide of type 1precollagen (P1NP) in serum were accreted in the HF group. Th17/Treg cell ratio in halofuginone-treated mice was 0.85 ± 0.05, and was significantly lower than that in PBS-treated mice, which was 1.51 ± 0.03. In addition, it showed that the Th17/Treg cell ratio was significantly and positively associated with osteoclasts, but was significantly and negatively associated with osteoblasts and bone remodeling. Halofuginone plays a critical role in the amelioration bone lesions in MM, as it can inhibit osteoclastogenesis and enhance osteoblastogenesis by regulating the Th17/Treg cell balance. [ABSTRACT FROM AUTHOR]

Prolyl‐tRNA synthetase (ProRS), belonging to the family of aminoacyl‐tRNA synthetases responsible for pairing specific amino acids with their respective tRNAs, is categorized into two distinct types: the eukaryote/archaeon‐like type (E‐type) and the prokaryote‐like type (P‐type). Notably, these types are specific to their corresponding cognate tRNAs. In an intriguing paradox, Thermus thermophilus ProRS (TtProRS) aligns with the E‐type ProRS but selectively charges the P‐type tRNAPro, featuring the bacterium‐specific acceptor‐stem elements G72 and A73. This investigation reveals TtProRS's notable resilience to the inhibitor halofuginone, a synthetic derivative of febrifugine emulating Pro‐A76, resembling the characteristics of the P‐type ProRS. Furthermore, akin to the P‐type ProRS, TtProRS identifies its cognate tRNA through recognition of the acceptor‐stem elements G72/A73, along with the anticodon elements G35/G36. However, in contrast to the P‐type ProRS, which relies on a strictly conserved R residue within the bacterium‐like motif 2 loop for recognizing G72/A73, TtProRS achieves this through a non‐conserved sequence, RTR, within the otherwise non‐interacting eukaryote‐like motif 2 loop. This investigation sheds light on the adaptive capacity of a typically conserved housekeeping enzyme to accommodate a novel substrate. [ABSTRACT FROM AUTHOR]

Glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is known to associated with fibrosis through its catalytic activity to produce prolyl-tRNA. Although its catalytic inhibitor halofuginone (HF) has been known to inhibit the TGF-β pathway as well as to reduce prolyl-tRNA production for the control of fibrosis, the underlying mechanism how EPRS1 regulates the TGF-β pathway was not fully understood. Here, we show a noncatalytic function of EPRS1 in controlling the TGF-β pathway and hepatic stellate cell activation via its interaction with TGF-β receptor I (TβRI). Upon stimulation with TGF-β, EPRS1 is phosphorylated by TGF-β-activated kinase 1 (TAK1), leading to its dissociation from the multi-tRNA synthetase complex and subsequent binding with TβRI. This interaction increases the association of TβRI with SMAD2/3 while decreases that of TβRI with SMAD7. Accordingly, EPRS1 stabilizes TβRI by preventing the ubiquitin-mediated degradation of TβRI. HF disrupts the interaction between EPRS1 and TβRI, and reduces TβRI protein levels, leading to inhibition of the TGF-β pathway. In conclusion, this work suggests the novel function of EPRS1 involved in the development of fibrosis by regulating the TGF-β pathway and the antifibrotic effects of HF by controlling both of EPRS1 functions.

Breast cancer presents significant challenges in oncology, particularly basal-like breast cancer (BLBC), which shows resistance to paclitaxel treatment. This study examines the efficacy of halofuginone (HF), a compound from traditional Chinese medicine, in mitigating this resistance by targeting the BRCA1/TGF-β signaling axis affecting epithelial-mesenchymal transition (EMT). Using analyses such as weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) enrichment from TCGA database RNA-seq data, along with transcriptomic sequencing and in vitro experiments, our findings demonstrate HF’s ability to modulate BRCA1 expression, impacting cell migration, invasion, and EMT in resistant BLBC cells. BRCA1 knockout experiments further reveal that HF’s effects on EMT can be reversed, underscoring the crucial role of the BRCA1-TGFBR2 axis in drug resistance. This study sheds light on HF’s mechanism in overcoming paclitaxel resistance and suggests new treatment avenues for BLBC, showcasing the potential integration of traditional Chinese medicine in modern therapeutic strategies.

In female dogs, the highest morbidity and mortality rates cancer are the result of mammary adenocarcinoma, which presents with metastases in the lung. Other than early surgical removal, however, no special methods are available to treat mammary adenocarcinoma. Because human breast cancer and canine mammary carcinoma share clinical characteristics and heterogeneity, the canine model is a suitable spontaneous tumor model for breast cancer in humans. In this study, the physical swelling method was used to prepare halofuginone-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles nano-thermosensitive hydrogels (HTPM-gel). Furthermore, HTPM-gel was investigated via characterization, morphology, properties such as swelling experiment and in vitro release with reflecting its splendid nature. Moreover, HTPM-gel was further examined its capability to anti-proliferation, anti-migration, and anti-invasion. Ultimately, HTPM-gel was investigated for its in vivo anticancer activity in the post-operative metastatic and angiogenic canine mammary carcinoma. HTPM-gel presented spherical under transmission electron microscope (TEM) and represented grid structure under scanning electron microscope (SEM), with hydrodynamic diameter (HD) of 20.25 ± 2.5 nm and zeta potential (ZP) of 15.10 ± 1.82 mV. Additionally, HTPM-gel own excellent properties comprised of pH-dependent swelling behavior, sustained release behavior. To impede the migration, invasion, and proliferation of CMT-U27 cells, we tested the efficacy of HTPM-gel. Evaluation of in vivo anti-tumor efficacy demonstrates HTPM-gel exhibit a splendid anti-metastasis and anti-angiogenic ability, with exhibiting ideal biocompatibility. Notably, HTPM-gel also inhibited the scar formation in the healing process after surgery. In summary, HTPM-gel exhibited anti-metastasis and anti-angiogenic and scar repair features. According to the results of this study, HTPM-gel has encouraging clinical potential to treat tumors with multifunctional hydrogel.

Background and purposeHalofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH).Experimental approachPatch-clamp experiments were conducted to examine the activity of voltage-dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2 ) for 4 weeks were used as model of HPH for in vivo experiments.Key resultsHalofuginone increased voltage-gated K+ (Kv ) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 μM) inhibited receptor-operated Ca2+ entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca2+ entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg-1 , for 2 weeks) partly reversed established PH in mice.Conclusion and implicationsHalofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor-operated and store-operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.

ObjectivePolyethylene glycol-modified gold nanostar particles (GNS-PEG) were constructed to investigate whether the degradation of extracellular matrix in triple-negative breast cancer could improve the tumor delivery of GNS-PEG and enhance the efficacy of photothermal therapy. MethodsGNS-PEG were constructed and characterized for physicochemical properties as well as photothermal properties. At the cellular level, the cytotoxicity of halofuginone (HF) and the effect of photothermal therapy were detected. Mouse model of triple negative breast cancer was established by subcutaneous inoculation of 4T1 cells in BALB/c nude mice. Five injections of HF were given via tail vein (HF group), and tumor sections were stained with Masson stain and immunohistochemical staining for transforming growth factor β1 (TGFβ1), α-smooth muscle actin (α-SMA) and CD31 to observe the effect of tumor stromal degradation. Five injections of HF via tail vein followed by GNS-PEG (HF+ GNS-PEG group) were applied to determine the content of gold in tumor tissues by inductively coupled plasma mass spectrometry. The tumor sites of the mice in the GNS-PEG and HF+ GNS-PEG groups were irradiated with NIR laser and the temperature changes were recorded with an IR camera. The tumour growth and weight changes of mice in each group were observed. Ki-67 immunohistochemical staining, TdT-mediated dUTP nick-end labeling and HE staining were performed on tumor tissue sections from each group to observe tumor proliferation, apoptosis and necrosis. HE staining was performed on heart, liver, spleen, lung and kidney tissues from each group to observe the morphological changes of cells. ResultsGNS-PEG nanoparticles showed a multi-branched structure with a particle size of 73.5±1.4 nm. The absorption peak of GNS was 810 nm, which is in the near infrared region. The photothermal conversion rate of GNS-PEG was up to 79.3%, and the photothermal effect could be controlled by the laser energy. HF has a concentration-dependent cytotoxicity, with a cell survival rate being as low as (22.8±2.6)% at HF concentration of up to 1 000 nmol/L. The photothermal effect of GNS-PEG was significant in killing tumor cells, with a cell survival rate of (32.7±5.2)% at the concentration of 25 pmol/L. The collagen area fraction, TGFβ1 integrated optical density and α-SMA integrated optical density in the tumor tissues of mice in the HF group were (2.1±0.2)%, 3.1±0.4 and 5.2±1.9, respectively, which were lower than those of the control group (all P<0.01), and the vessel diameter was 8.6±2.9 μm, which was higher than that of the control group (P<0.05). In the HF+ GNS-PEG group, the concentration of gold in tissues was 52.4 μg/g, higher than that in the GNS-PEG group (15.9 μg/g, P<0.05). After laser irradiation, the temperature of the tumor site in the HF+ GNS-PEG group was significantly higher than that in the GNS-PEG group. At the 4th minute, the temperatures of the tumor site in the GNS-PEG and HF+ GNS-PEG groups were 51.5 ℃ and 57.7 ℃ respectively; the tumor volume in the HF+ GNS-PEG group was effectively suppressed. The body weights of the mice in each group did not change significantly during the monitoring period. No significant abnormalities were observed in the main organs of the mice in the GNS-PEG group, but some hepatocytes in the HF and HF+ GNS-PEG groups showed edema and degeneration. ConclusionThe remodeling of extracellular matrix in triple-negative breast cancer could significantly improve the intratumoral delivery of GNS-PEG and thus achieve better photothermal therapy effect. [ABSTRACT FROM AUTHOR]

The enriched collagens in the extracellular matrix (ECM) of breast cancer substantially impede drug delivery. Halofuginone (HF), a potent antifibrotic agent, was effective to deplete the collagens and remodel the ECM by inhibiting the TGFβ pathway. However, the application of HF was hindered by its strong liver toxicity. Herein, mesoporous platinum (mPt) nanoparticles were constructed to load HF as theranostic nanoplatforms. mPt had a uniform spherical structure with a diameter of 79.83 ± 6.97 nm and an average pore diameter of 20 nm and exhibited good photothermal conversion efficiency of 62.4%. The obtained HF‐loaded nanoplatform (PEG@mPt‐HF) showed enhanced cytotoxicity through the combination of photothermal therapy and the anti‐TGFβ effect induced by HF. The animal imaging and histochemical assays confirmed the PEG@mPt‐HF could efficiently deliver HF to tumors (monitored by CT) and remodel the ECM by TGFβ pathway inhibition, which resulted in increased anti‐cancer efficacy. Importantly, the liver toxicity observed in HF‐treated mice was negligible in those treated by PEG@mPt‐HF. Overall, this study designed a theranostic nanoplatform to remodel the ECM with remarkably reduced systematic toxicity and enhance the therapeutic efficacy through combination treatment. [ABSTRACT FROM AUTHOR]

The antiparasitic drug halofuginone is important for controlling apicomplexan parasites. However, the occurrence of halofuginone resistance is a major obstacle for it to the treatment of apicomplexan parasites. Current studies have identified the molecular marker and drug resistance mechanisms of halofuginone in Plasmodium falciparum. In this study, we tried to use transcriptomic data to explore resistance mechanisms of halofuginone in apicomplexan parasites of the genus Eimeria (Apicomplexa: Eimeriidae). After halofuginone treatment of E. tenella parasites, transcriptome analysis was performed using samples derived from both resistant and sensitive strains. In the sensitive group, DEGs associated with enzymes were significantly downregulated, whereas the DNA damaging process was upregulated after halofuginone treatment, revealing the mechanism of halofuginone-induced parasite death. In addition, 1,325 differentially expressed genes (DEGs) were detected between halofuginone resistant and sensitive strains, and the DEGs related to translation were significantly downregulated after halofuginone induction. Overall, our results provide a gene expression profile for further studies on the mechanism of halofuginone resistance in E. tenella. [ABSTRACT FROM AUTHOR]

The crystal structure of one form of halofuginone hydrobromide has been solved and refined using synchrotron X-ray powder diffraction data, and optimized using density functional theory techniques. Halofuginone hydrobromide crystallizes in space group P2 1 (#4) with a = 8.87398(13), b = 14.25711(20), c = 15.0153(3) Å, β = 91.6867(15)°, V = 1898.87(4) Å3, and Z = 4. The crystal structure consists of alternating layers (parallel to the ab -plane) of planar and nonplanar portions of the cations. N–H⋯Br and O–H⋯Br hydrogen bonds link the protonated piperidine rings and bromide anions into a two-dimensional network parallel to the ab -plane. The powder pattern has been submitted to ICDD for inclusion in the Powder Diffraction File™ (PDF®). [ABSTRACT FROM AUTHOR]

Abstract The enriched collagens in the extracellular matrix (ECM) of breast cancer substantially impede drug delivery. Halofuginone (HF), a potent antifibrotic agent, was effective to deplete the collagens and remodel the ECM by inhibiting the TGFβ pathway. However, the application of HF was hindered by its strong liver toxicity. Herein, mesoporous platinum (mPt) nanoparticles were constructed to load HF as theranostic nanoplatforms. mPt had a uniform spherical structure with a diameter of 79.83 ± 6.97 nm and an average pore diameter of 20 nm and exhibited good photothermal conversion efficiency of 62.4%. The obtained HF‐loaded nanoplatform (PEG@mPt‐HF) showed enhanced cytotoxicity through the combination of photothermal therapy and the anti‐TGFβ effect induced by HF. The animal imaging and histochemical assays confirmed the PEG@mPt‐HF could efficiently deliver HF to tumors (monitored by CT) and remodel the ECM by TGFβ pathway inhibition, which resulted in increased anti‐cancer efficacy. Importantly, the liver toxicity observed in HF‐treated mice was negligible in those treated by PEG@mPt‐HF. Overall, this study designed a theranostic nanoplatform to remodel the ECM with remarkably reduced systematic toxicity and enhance the therapeutic efficacy through combination treatment.

Activating transcription factor 4 (ATF4) is involved in muscle atrophy through the overexpression of some atrogenes. However, it also controls the transcription of genes involved in muscle homeostasis maintenance. Here, we explored the effect of ATF4 activation by the pharmacological molecule halofuginone during hindlimb suspension (HS)-induced muscle atrophy. Firstly, we reported that periodic activation of ATF4-regulated atrogenes (Gadd45a, Cdkn1a, and Eif4ebp1) by halofuginone was not associated with muscle atrophy in healthy mice. Secondly, halofuginone-treated mice even showed reduced atrophy during HS, although the induction of the ATF4 pathway was identical to that in untreated HS mice. We further showed that halofuginone inhibited transforming growth factor-β (TGF-β) signalling, while promoting bone morphogenetic protein (BMP) signalling in healthy mice and slightly preserved protein synthesis during HS. Finally, ATF4-regulated atrogenes were also induced in the atrophy-resistant muscles of hibernating brown bears, in which we previously also reported concurrent TGF-β inhibition and BMP activation. Overall, we show that ATF4-induced atrogenes can be uncoupled from muscle atrophy. In addition, our data also indicate that halofuginone can control the TGF-β/BMP balance towards muscle mass maintenance. Whether halofuginone-induced BMP signalling can counteract the effect of ATF4-induced atrogenes needs to be further investigated and may open a new avenue to fight muscle atrophy. Finally, our study opens the way for further studies to identify well-tolerated chemical compounds in humans that are able to fine-tune the TGF-β/BMP balance and could be used to preserve muscle mass during catabolic situations. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial regions, with a high rate of metastasis. Cancerassociated fibroblasts (CAFs) play critical roles in tumor growth, metastasis and invasion, making them attractive therapeutic targets for cancer treatment. As an old anti-coccidiosis drug for poultry, Halofuginone (HF) has also been reported to possess anti-fibrosis and anti-cancer activities in the recent decades. However, whether it works by targeting CAFs in OSCC, and the mechanisms involved remain unclear. In the present study, we observed HF dose-dependently inhibits OSCC-derived CAF viability and proliferation. Meanwhile, HF decreased the expressions of α-SMA, FSP-1 and PDGFRβ, markers of the malignant phenotype of CAFs, both at mRNA and protein levels. Furthermore, functional studies demonstrated that HF dramatically attenuates the promotion effect of CAFs on OSCC cell migration and invasion. Mechanistically, the inhibition of MMP2 secretion and the upstream TGF-ß/Smad2/3 signaling pathway played an important role in these processes. In the orthotopic transplanted tongue carcinoma in mice model, we confirmed that HF administration inhibited tumor growth and lymph node metastasis (LNM) with reduced CAF population, MMP2 expression and collagen deposition in tumor. Altogether, these results indicate that HF can inhibit the migration and invasion of OSCC by targeting CAFs, which will provide new ideas for the treatment of OSCC. [ABSTRACT FROM AUTHOR]

The present study aimed to explore the effects of antifibrotic agent halofuginone on uterine leiomyomas (ULs) cells. The survival of the uterine smooth muscle (UtSMC) cells and UL ELT3 cells were measured. Flow cytometry was used to assess the cell cycle distribution and apoptosis. Effects of halofuginone on the state of AKT/mTOR pathway were evaluated. Xenograft animal model was applied to explore the effects of halofuginone in vivo. Halofuginone inhibited the proliferation of ELT3 cells dose-dependently without obvious influence on UtSMC cells. Halofuginone suppressed cell cycle progression and promoted apoptosis of ELT3 cells dose-dependently. Also, p-AKT/AKT and p-p70S6/p70S6 were significantly lowered after treatment with 20 nM halofuginone. Additionally, halofuginone reduced ELT3 tumor growth in xenograft tumor animal model. The present study illustrates that halofuginone inhibits cell proliferation of ULs with low side effects on normal smooth muscle cells, and AKT/mTOR signaling pathway was inactivated meanwhile. [ABSTRACT FROM AUTHOR]

• The global prevalence of parasitic protozoan diseases remains a significant public health challenge. • Halofuginone, a plant-derived compound, has beneficial roles in veterinary and human diseases. • The therapeutic potential of halofuginone as an antiprotozoal is summarized. • The primary mode of action of halofuginone via inhibition of prolyl-tRNA synthetase is discussed. Halofuginone is a clinically active derivative of febrifugine that was first isolated from the Chinese herb Dichroa febrifuga. The beneficial biological effects of halofuginone on various diseases including parasitic diseases, cancer, fibrosis, and autoimmune disorders have been investigated. Halofuginone has reduced toxic side effects when compared to febrifugine, an advantage that has led to the commercial availability of halofuginone-based antiparasitic drugs for animal use, and to human clinical trials for the treatment of tumors and fibrosis. This review summarizes advances in determining the mechanism of action of halofuginone, focusing on its antiparasitic role in malaria, cryptosporidiosis, coccidiosis, toxoplasmosis, and leishmaniasis. We discuss mechanistic insights into halofuginone's primary mode of action which involves inhibition of the prolyl-tRNA synthetase enzyme, which is crucial in protein synthesis. Halofuginone exemplifies the untapped wealth of plant-derived compounds in disease therapeutics. [ABSTRACT FROM AUTHOR]

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti-P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl-terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6' region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti-malarial agents in the future., (© 2024 Wiley-VCH GmbH.)

Cancer is still considered a “hopeless case”, which seriously endangers human health. Therefore, it is of great significance to research and develop novel anticancer drugs. Active natural products play an important role in anticancer drug discovery by providing lead compounds. Halofuginone (HF), as a derivative of febrifugine, an alkaloid originally isolated from the plant Dichroa febrifuga, is used for the prevention and treatment of coccidiosis and malaria in poultry. In recent years, HF has shown excellent anticancer activity and great clinical potential for treating various cancers. Despite enormous progress, a thorough elaboration of the proposed mechanism and mode of action is absent. Herein, we provide a comprehensive review of the most recent anticancer studies reported for HF and a rationale for further exploring the potential application of HF in overcoming cancer in the future.

Abstract Objectives To evaluate antifibrotic effects of corticosteroids and halofuginone, a small molecule inhibitor of Smad3, in an ovine model of vocal fold (VF) injury. Methods Thirty sheep, using a paired study design, underwent controlled right VF injury by biopsy and then were treated with either no treatment, oral dexamethasone, intralesional triamcinolone, or oral halofuginone. Larynges were evaluated for histological evidence of fibrosis, immunohistochemical presence of Smad3, and vibratory parameters. Outcomes were compared across treatment groups. Results Following injury, VF collagen density decreased in both halofuginone‐treated and dexamethasone‐treated sheep but not in triamcinolone treated sheep. A significant difference was noted between halofuginone and triamcinolone treated sheep (27.8% vs 37%, P = .017). Elastin was preserved postinjury by halofuginone treatment in contrast with all steroid treated animals where significant loss of elastin was noted (P

The synthesis of both enantiomers of a key intermediate in the synthesis of halofuginone was accomplished by a Candida antarctica lipase B (CAL-B)-catalyzed kinetic resolution of the corresponding racemate. When the resolution was carried out in the versatile solvent cyclopentyl methyl ether (CPME) using p-chlorophenylbutyrate (PCPB) as the acylating reagent, the highest enantiomeric ratio (E) values were measured, and highly enantioenriched (95% ee) compounds could be obtained in a single iteration. As an example, one of the two enantiomers was used as a starting material to prepare (+)-halofuginone in a three-step procedure.

[Display omitted] • Halofuginone-silver thermosensitive nano-in-situ-hydrogels (HTPM&AgNPs-gel) were successfully fabricated using the physical swelling technique. • HTPM&AgNPs-gel had excellent properties, including injectable, ductile, thermosensitive with thermal stability, excellent properties including syringeability, swelling behavior, a sustained release effect, and favorable hemocompatibility. • HTPM&AgNPs-gel also inhibited lung metastasis induced by residual tumor tissue after surgery and further blocked angiogenesis-related inflammatory responses compared with commercial thermosensitive nanohydrogels. • The suppression of inflammation by interdicting the blood vessels adjoining the tumor and inhibiting angiogenesis is a potential strategy to attenuate the recurrence and metastasis of TNBC. • HTPM&AgNPs-gel is a promising anticancer agent for TNBC as a local treatment. Postoperative distant metastasis and high recurrence rate causes a dilemma in treating triple-negative breast cancer (TNBC) owing to its unforeseeable invasion into various organs or tissues. The wealth of nutrition provided by vascular may facilitate the proliferation and angiogenesis of cancer cells, which further enhance the rates of postoperative metastasis and recurrence. Chemotherapy, as a systemic postoperative adjuvant therapy, is generally applied to diminish recurrence and metastasis of TNBC. Herein, an halofuginone-silver nano thermosensitive hydrogel (HTPM&AgNPs-gel) was prepared via a physical swelling method. The in vitro anticancer efficacy of HTPM&AgNPs-gel was analyzed by investigating cell proliferation, migration, invasion, and angiogenesis capacity. Furthermore, the in vivo anti-cancer activity of HTPM&AgNPs-gel was further appraised through the tumor suppression, anti-metastatic, anti-angiogenic, and anti-inflammatory ability. The optimized HTPM&AgNPs-gel, a thermosensitive hydrogel, showed excellent properties, including syringeability, swelling behavior, and a sustained release effect without hemolysis. In addition, HTPM&AgNPs-gel was confirmed to effectively inhibit the proliferation, migration, invasion, and angiogenesis of MDA-MB-231 cells. An evaluation of the in vivo anti-tumor efficacy demonstrated that HTPM&AgNPs-gel showed a stronger tumor inhibition rate (68.17%) than did HTPM-gel or AgNPs-gel used alone and exhibited outstanding biocompatibility. Notably, HTPM&AgNPs-gel also inhibited lung metastasis induced by residual tumor tissue after surgery and further blocked angiogenesis-related inflammatory responses. Taken together, the suppression of inflammation by interdicting the blood vessels adjoining the tumor and inhibiting angiogenesis is a potential strategy to attenuate the recurrence and metastasis of TNBC. HTPM&AgNPs-gel is a promising anticancer agent for TNBC as a local postoperative treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] • This study discovered the EMT-associated Blue module. • This study identified 230 genes that were differentially expressed and mediated by TGF-β. • This study confirms that Halofuginone targets BRCA1, which is crucial. • The present study elucidates the effects of Halofuginone on the process of epithelial-mesenchymal transition (EMT) in basal-like breast cancer (BLBC) cells. • This study provides new treatment strategies for breast cancer. Breast cancer presents significant challenges in oncology, particularly basal-like breast cancer (BLBC), which shows resistance to paclitaxel treatment. This study examines the efficacy of halofuginone (HF), a compound from traditional Chinese medicine, in mitigating this resistance by targeting the BRCA1/TGF-β signaling axis affecting epithelial-mesenchymal transition (EMT). Using analyses such as weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) enrichment from TCGA database RNA-seq data, along with transcriptomic sequencing and in vitro experiments, our findings demonstrate HF's ability to modulate BRCA1 expression, impacting cell migration, invasion, and EMT in resistant BLBC cells. BRCA1 knockout experiments further reveal that HF's effects on EMT can be reversed, underscoring the crucial role of the BRCA1-TGFBR2 axis in drug resistance. This study sheds light on HF's mechanism in overcoming paclitaxel resistance and suggests new treatment avenues for BLBC, showcasing the potential integration of traditional Chinese medicine in modern therapeutic strategies. [ABSTRACT FROM AUTHOR]

Anaplastic thyroid carcinoma (ATC) is recognized as the most aggressive and malignant form of thyroid cancer, underscoring the critical need for effective therapeutic strategies to curb its progression and improve patient prognosis. Halofuginone (HF), a derivative of febrifugine, has displayed antitumor properties across various cancer types. However, there is a paucity of published research focused on the potential of HF to enhance the clinical efficacy of treating ATC. In this study, we thoroughly investigated the antitumor effects and mechanisms of HF in ATC, aiming to discover lead compounds for treating ATC and reveal novel therapeutic targets for ATC tumors. A series of assays, including CCK8, colony formation, tumor xenograft models, and ATC tumor organoid experiments, were conducted to evaluate the anticancer properties of HF both in vitro and in vivo. Techniques such as drug affinity responsive target stability (DARTS), western blot, immunofluorescence, and immunohistochemistry were employed to pinpoint HF target proteins within ATC. Furthermore, we harnessed the GEPIA and GEO databases and performed immunohistochemistry to validate the therapeutic potential of the glutamyl-prolyl-tRNA-synthetase (EPRS)- activating transcription factor 4 (ATF4)- type I collagen (COLI) pathway axis in the context of ATC. The study also incorporated RNA sequencing analysis, confocal imaging, and flow cytometry to delve into the molecular mechanisms of HF in ATC. HF exhibited a substantial inhibitory impact on cell proliferation in vitro and on tumor growth in vivo. The DARTS results highlighted HF's influence on EPRS within ATC cells, triggering an amino acid starvation response (AASR) by suppressing EPRS expression, consequently leading to a reduction in COLI expression in ATC cells. The introduction of proline mitigated the effect of HF on ATF4 and COLI expression, indicating that the EPRS-ATF4-COLI pathway axis was a focal target of HF in ATC. Analysis of the expression levels of the EPRS, ATF4, and COLI proteins in thyroid tumors, along with an examination of the relationship between COLI expression and thyroid tumor stage, revealed that HF significantly inhibited the growth of ATC tumor organoids, demonstrating the therapeutic potential of targeting the EPRS-ATF4-COLI pathway axis in ATC. RNA sequencing analysis revealed significant differences in the pathways associated with metastasis and apoptosis between control and HF-treated cells. Transwell assays and flow cytometry experiments provided evidence of the capacity of HF to impede cell migration and induce apoptosis in ATC cells. Furthermore, HF hindered cell metastasis by suppressing the epithelial-mesenchymal transition (EMT) pathway, acting through the inhibition of FAK-AKT-NF-κB/Wnt-β-catenin signaling and restraining angiogenesis via the VEGF pathway. HF also promoted apoptosis through the mitochondrial apoptotic pathway. This study provided inaugural evidence suggesting that HF could emerge as a promising therapeutic agent for the treatment of ATC. The EPRS-ATF4-COLI pathway axis stood out as a prospective biomarker and therapeutic target for ATC. [Display omitted] [ABSTRACT FROM AUTHOR]

Halofuginone (HFG) is a widely used anti-coccidiosis drug in animal husbandry. There is an urgent need for a rapid detection method to detect HFG residues in chicken and beef to ensure human health. In this study, suitable HFG haptens were selected using computer simulation, and a highly sensitive monoclonal antibody (mAb) was developed with the 50%-inhibitory concentration (IC 50) of 0.66 ng/mL. Then, gold nanoparticle-based lateral flow immunochromatographic (ICA) strips for HFG detection were produced. Under optimization conditions, the ICA strips exhibited outstanding performance with a visual limit of detection (vLOD) of 50 μg/kg and 5 μg/kg, and cut-off values of 100 μg/kg and 10 μg/kg, for chicken and beef samples, respectively. In addition, the reliability of strips was verified by enzyme linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Therefore, the developed strip can be used as an effective tool for HFG residues screening in chicken and beef samples. [ABSTRACT FROM AUTHOR]

In this study, a Janus nanoplatform consisting of periodic mesoporous organosilica-coated platinum nanoplatforms and halofuginone was developed to deplete the tumor stroma and synergistically treat the tumor. [Display omitted] Dense tumor stroma is the physiological barrier in drug delivery that prevents anticancer drugs from entering the tumor, thereby seriously limiting the drugs' therapeutic effect. In this study, a Janus nanoplatform consisting of periodic mesoporous organosilica-coated platinum nanoplatforms (JPMO-Pt) and anti-stroma drug halofuginone (HF) (denoted as JPMO-Pt-HF), was developed to deplete the tumor stroma and synergistically treat breast cancer in BALB/c mice. The prepared JPMO-Pt had a uniform size of 245 nm, a good dispersion, an excellent in vitro and in vivo biocompatibility, and a high loading capacity for HF (up to 50 μg/mg). The antitumor experiments showed that the survival rate of 4 T1 cells exhibited an obvious downward trend when the cells were incubated with the JPMO-Pt-HF and irradiated with 808 nm laser. Moreover, the cell survival rate was only about 10% at 48 h when the HF concentration was 2.0 μg/mL. Notably, JPMO-Pt-HF under irradiation had an excellent synergistic therapeutic effect on tumor cells. In vivo antitumor experiment further showed that the JPMO-Pt-HF, in combination with laser irradiation, could minimize tumor growth, showing significantly better effects than those observed for the case of monotherapy involving photothermal therapy (PTT) (152 vs. 670 mm3, p < 0.0001) and HF (152 vs. 419 mm3, p = 0.0208). In addition, immunohistochemistry of tumor tissues indicated that JPMO-Pt-HF obviously reduced the relative collagen and α-smooth muscle actin (α-SMA) area fraction. Taken together, this research designs a new platform that not only possesses the ability to degrade the tumor matrix but also combines PTT and chemotherapeutic effects, and holds promise for effective tumor treatment. [ABSTRACT FROM AUTHOR]

Solid tumors are characterized by abundant extracellular matrix originating from cancer-associated fibroblasts (CAFs). High collagen content can trigger the collapse of vascular system in the tumor and form physical barrier that eventually impedes the penetration of drug particles and cytotoxic immune cells. Moreover, CAFs is able to promote the enrichment of tumor-associated macrophages (TAMs) and differentiation of myeloid-derived suppressor cells (MDSCs) that work in concert to develop a highly immunosuppressive tumor microenvironment (TME). In this study, we investigated if halofuginone, an antifibrotic drug, can augment the therapeutic effects of oncolytic vesicular stomatitis virus (VSV). The results revealed that halofuginone significantly disrupts the collagen network in tumors and promotes the distribution of VSV and infiltration of CD8+ T cells (p < 0.0001). Combined treatment of VSV and halofuginone also modulates the immunosuppressive TME via deletion of TAM, MDSCs, and regulatory T cells (Tregs). Collectively, the combination therapy remarkably inhibits the tumor growth in multiple murine models and prolongs survival of mice. The results demonstrate the clinical potential of halofuginone in combination with oncolytic virus. [ABSTRACT FROM AUTHOR]

Activating transcription factor 4 (ATF4) is involved in muscle atrophy through the overexpression of some atrogenes. However, it also controls the transcription of genes involved in muscle homeostasis maintenance. Here, we explored the effect of ATF4 activation by the pharmacological molecule halofuginone during hindlimb suspension (HS)-induced muscle atrophy. Firstly, we reported that periodic activation of ATF4-regulated atrogenes (Gadd45a, Cdkn1a, and Eif4ebp1) by halofuginone was not associated with muscle atrophy in healthy mice. Secondly, halofuginone-treated mice even showed reduced atrophy during HS, although the induction of the ATF4 pathway was identical to that in untreated HS mice. We further showed that halofuginone inhibited transforming growth factor-β (TGF-β) signalling, while promoting bone morphogenetic protein (BMP) signalling in healthy mice and slightly preserved protein synthesis during HS. Finally, ATF4-regulated atrogenes were also induced in the atrophy-resistant muscles of hibernating brown bears, in which we previously also reported concurrent TGF-β inhibition and BMP activation. Overall, we show that ATF4-induced atrogenes can be uncoupled from muscle atrophy. In addition, our data also indicate that halofuginone can control the TGF-β/BMP balance towards muscle mass maintenance. Whether halofuginone-induced BMP signalling can counteract the effect of ATF4-induced atrogenes needs to be further investigated and may open a new avenue to fight muscle atrophy. Finally, our study opens the way for further studies to identify well-tolerated chemical compounds in humans that are able to fine-tune the TGF-β/BMP balance and could be used to preserve muscle mass during catabolic situations.

Prolyl-tRNA synthetase (ProRS), belonging to the family of aminoacyl-tRNA synthetases responsible for pairing specific amino acids with their respective tRNAs, is categorized into two distinct types: the eukaryote/archaeon-like type (E-type) and the prokaryote-like type (P-type). Notably, these types are specific to their corresponding cognate tRNAs. In an intriguing paradox, Thermus thermophilus ProRS (TtProRS) aligns with the E-type ProRS but selectively charges the P-type tRNA Pro , featuring the bacterium-specific acceptor-stem elements G72 and A73. This investigation reveals TtProRS's notable resilience to the inhibitor halofuginone, a synthetic derivative of febrifugine emulating Pro-A76, resembling the characteristics of the P-type ProRS. Furthermore, akin to the P-type ProRS, TtProRS identifies its cognate tRNA through recognition of the acceptor-stem elements G72/A73, along with the anticodon elements G35/G36. However, in contrast to the P-type ProRS, which relies on a strictly conserved R residue within the bacterium-like motif 2 loop for recognizing G72/A73, TtProRS achieves this through a non-conserved sequence, RTR, within the otherwise non-interacting eukaryote-like motif 2 loop. This investigation sheds light on the adaptive capacity of a typically conserved housekeeping enzyme to accommodate a novel substrate., (© 2024 The Protein Society.)

Lung cancer is the leading cause of cancer death worldwide. Cisplatin is the major DNA-damaging anticancer drug that cross-links the DNA in cancer cells, but many patients inevitably develop resistance with treatment. Identification of a cisplatin sensitizer might postpone or even reverse the development of cisplatin resistance. Halofuginone (HF), a natural small molecule isolated from Dichroa febrifuga, has been found to play an antitumor role. In this study, we found that HF inhibited the proliferation, induced G0/G1 phase arrest, and promoted apoptosis in lung cancer cells in a dose-dependent manner. To explore the underlying mechanism of this antitumor effect of halofuginone, we performed RNA sequencing to profile transcriptomes of NSCLC cells treated with or without halofuginone. Gene expression profiling and KEGG analysis indicated that PI3K/AKT and MAPK signaling pathways were top-ranked pathways affected by halofuginone. Moreover, combination of cisplatin and HF revealed that HF could sensitize the cisplatin-resistant patient-derived lung cancer organoids and lung cancer cells to cisplatin treatment. Taken together, this study identified HF as a cisplatin sensitizer and a dual pathway inhibitor, which might provide a new strategy to improve prognosis of patients with cisplatin-resistant lung cancer.

Objectives: To evaluate antifibrotic effects of corticosteroids and halofuginone, a small molecule inhibitor of Smad3, in an ovine model of vocal fold (VF) injury. Methods: Thirty sheep, using a paired study design, underwent controlled right VF injury by biopsy and then were treated with either no treatment, oral dexamethasone, intralesional triamcinolone, or oral halofuginone. Larynges were evaluated for histological evidence of fibrosis, immunohistochemical presence of Smad3, and vibratory parameters. Outcomes were compared across treatment groups. Results: Following injury, VF collagen density decreased in both halofuginone‐treated and dexamethasone‐treated sheep but not in triamcinolone treated sheep. A significant difference was noted between halofuginone and triamcinolone treated sheep (27.8% vs 37%, P =.017). Elastin was preserved postinjury by halofuginone treatment in contrast with all steroid treated animals where significant loss of elastin was noted (P <.05). Smad3 staining was up‐regulated at all injury sites compared to normal left VFs however halofuginone and dexamethasone treatment reduced Smad3 activity significantly whereas triamcinolone treatment did not (P <.05). Ex‐vivo stroboscopic evaluation demonstrated mucosal wave in all excised larynges with a normalized glottal gap less than 3, suggesting adequate glottal closure. Conclusions: VF injury in an ovine model results in a wound response able to be modified by Smad3 inhibitor, halofuginone, with benefit to vibratory function. Halofuginone treated sheep demonstrated reduced collagenization of lamina propria with greater elastin density after injury, than sheep treated with either steroid medication. These data support this pathway as a suitable target for manipulation to prevent or reverse fibrosis in the glottis and restore voice quality. Level of Evidence: NA. [ABSTRACT FROM AUTHOR]

Prostate cancer is known to have a relatively good prognosis, but long-term hormone therapy can lead to castration-resistant prostate cancer (CRPC). Cabazitaxel, a second-generation taxane, has been used for the CRPC treatment, but its tolerance is an urgent problem to be solved. In this study, to elucidate the acquisition mechanism of the cabazitaxel-resistance, we established cabazitaxel-resistant prostate cancer 22Rv1 (Cab-R) cells, which exhibited ∼sevenfold higher LD50 against cabazitaxel than the parental 22Rv1 cells. Cab-R cells showed marked increases in nuclear accumulation of NF-E2 related factor 2 (Nrf2) and expression of Nrf2-inducible antioxidant enzymes compared to 22Rv1 cells, suggesting that Nrf2 signalling is homeostatically activated in Cab-R cells. The cabazitaxel sensitivity of Cab-R cells was enhanced by silencing of Nrf2, and that of 22Rv1 cells was reduced by activation of Nrf2. Halofuginone (HF) has been recently identified as a potent Nrf2 synthetic inhibitor, and its treatment of Cab-R cells not only suppressed the Nrf2 signalling by decreasing both nuclear and cytosolic Nrf2 protein levels, but also significantly augmented the cabazitaxel sensitivity. Thus, inhibition of Nrf2 signalling may be effective in overcoming the cabazitaxel resistance in prostate cancer cells. [ABSTRACT FROM AUTHOR]

Abstract Background Helminth infections affect ~ 60% of the human population that lives in tropical and subtropical regions worldwide. These infections result in diseases like schistosomiasis, lymphatic filariasis, river blindness and echinococcosis. Here we provide a comprehensive computational analysis of the aminoacyl tRNA synthetase (aaRS) enzyme family from 27 human-infecting helminths. Our analyses support the idea that several helminth aaRSs can be targeted for drug repurposing or for development of new drugs. For experimental validation, we focused on Onchocerciasis (also known as “river blindness”), a filarial vector-borne disease that is prevalent in Africa and Latin America. We show that halofuginone (HF) can act as a potent inhibitor of Onchocerca volvulus prolyl tRNA synthetase (OvPRS). Results The conserved enzyme family of aaRSs has been validated as druggable targets in numerous eukaryotic parasites. We thus embarked on assessing aaRSs from the genomes of 27 helminths that cause infections in humans. In order to delineate the distribution of aaRSs per genome we utilized Hidden Markov Models of aaRS catalytic domains to identify all orthologues. We note that Fasciola hepatica genome encodes the highest number of aaRS-like proteins (69) whereas Taenia asiatica has the lowest count (32). The number of genes for any particular aaRS-like protein varies from 1 to 8 in these 27 studied helminths. Sequence alignments of helminth-encoded lysyl, prolyl, leucyl and threonyl tRNA synthetases suggest that various known aaRS inhibitors like Cladosporin, Halofuginone, Benzoborale and Borrelidin may be of utility against helminths. The recombinantly expressed Onchocerca volvulus PRS was used as proof of concept for targeting aaRS with drug-like molecules like HF. Conclusions Systematic analysis of unique subdomains within helminth aaRSs reveals the presence of a number of non-canonical domains like PAC3, Utp-14, Pex2_Pex12 fused to catalytic domains in the predicted helminth aaRSs. We have established a platform for biochemical validation of a large number of helminth aaRSs that can be targeted using available inhibitors to jump-start drug repurposing against human helminths.

A highly sensitive, specific, and rapid immunochromatographic assay for halofuginone (HFG) has been developed. A well-characterized immunogenic conjugate of HFG with bovine serum albumin was produced using the N,N″-Carbonyldiimidazole method. A sensitive monoclonal antibody specific for HFG was then generated by immunizing BALB/c mic with the HFG conjugate. The antigen, HFG-CDI-ovalbumin and goat anti-mouse IgG were attached to a nitrocellulose membrane as the test line and the control line, respectively to produce an immunochromatographic test strip for the rapid detection of HFG in milk. Under optimized conditions, the detection limits of the semi-quantitative test strip for HFG were as low as 50 ng/mL in 0.01 M PBS (pH 7.4) and 100 ng/mL in milk. All the results were obtained within 5 min. The newly developed immunochromatographic assay provides a sensitive, rapid, and specific procedure for on-site screening for HFG.

Keloids are characterized by increased deposition of fibrous tissue in the skin and subcutaneous tissue following an abnormal wound healing process. Although keloid etiology is yet to be fully understood, fibroblasts are known to be key players in its development. Here we analyze the antifibrotic mechanisms of Halofuginone (HF), a drug reportedly able to inhibit the TGF-β1-Smad3 pathway and to attenuate collagen synthesis, in an in-vitro keloid model using patient-derived Keloid Fibroblasts (KFs) isolated from fibrotic tissue collected during the “Scar Wars” clinical study (NCT NCT03312166). TGF-β1 was used as a pro-fibrotic agent to stimulate fibroblasts response under HF treatment. The fibrotic related properties of KFs, including survival, migration, proliferation, myofibroblasts conversion, ECM synthesis and remodeling, were investigated in 2D and 3D cultures. HF at 50 nM concentration impaired KFs proliferation, and decreased TGF-β1-induced expression of α-SMA and type I procollagen production. HF treatment also reduced KFs migration, prevented matrix contraction and increased the metallo-proteases/inhibitors (MMP/TIMP) ratio. Overall, HF elicits an anti-fibrotic contrasting the TGF-β1 stimulation of KFs, thus supporting its therapeutic use for keloid prevention and management.

Aims: Transforming growth factor‐β (TGF‐β) signalling is thought to contribute to the remodelling of extracellular matrix (ECM) of skeletal muscle and to functional decline in patients with muscular dystrophies. We wanted to determine the role of TGF‐β‐induced ECM remodelling in dystrophic muscle. Methods: We experimentally induced the pathological hallmarks of severe muscular dystrophy by mechanically overloading the plantaris muscle in mice. Furthermore, we determined the role of TGF‐β signalling on dystrophic tissue modulation and on muscle function by (i) overloading myostatin knockout (Mstn−/−) mice and (ii) by additional pharmacological TGF‐β inhibition via halofuginone. Results: Transcriptome analysis of overloaded muscles revealed upregulation predominantly of genes associated with ECM, inflammation and metalloproteinase activity. Histology revealed in wild‐type mice signs of severe muscular dystrophy including myofibres with large variation in size and internalized myonuclei, as well as increased ECM deposition. At the same time, muscle weight had increased by 208% and muscle force by 234%. Myostatin deficiency blunted the effect of overload on muscle mass (59% increase) and force (76% increase), while having no effect on ECM deposition. Concomitant treatment with halofuginone blunted overload‐induced muscle hypertrophy and muscle force increase, while reducing ECM deposition and increasing myofibre size. Conclusions: ECM remodelling is associated with an increase in muscle mass and force in overload‐modelled dystrophic muscle. Lack of myostatin is not advantageous and inhibition of ECM deposition by halofuginone is disadvantageous for muscle plasticity in response to stimuli that induce dystrophic muscle. [ABSTRACT FROM AUTHOR]

Currently available drugs being used to treat leishmaniasis have several shortcomings, including high toxicity, drug administration that requires hospitalization, and the emergence of parasite resistance against clinically used drugs. As a result, there is a dire need for the development of new antileishmanial drugs that are safe, affordable, and efficient. In this study, two new series of synthesized quinazolinone derivatives were investigated as potential future antileishmanial agents, by assessing their activities against the Leishmania (L.) donovani and L. major species. The cytotoxicity profiles of these derivatives were assessed in vitro on Vero cells. The compounds were found to be safer and without any toxic activities against mammalian cells, compared to the reference drug, halofuginone, a clinical derivative of febrifugine. However, they had demonstrated poor antileishmanial growth inhibition efficacies. The two compounds that had been found the most active were the mono quinazolinone 2d and the bisquinazolinone 5b with growth inhibitory efficacies of 35% and 29% for the L. major and L. donovani 9515 promastigotes, respectively. These outcomes had suggested structural redesign, inter alia the inclusion of polar groups on the quinazolinone ring, to potentially generate novel quinazolinone derivatives, endowed with effective antileishmanial potential. [ABSTRACT FROM AUTHOR]

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In female dogs, the highest morbidity and mortality rates cancer are the result of mammary adenocarcinoma, which presents with metastases in the lung. Other than early surgical removal, however, no special methods are available to treat mammary adenocarcinoma. Because human breast cancer and canine mammary carcinoma share clinical characteristics and heterogeneity, the canine model is a suitable spontaneous tumor model for breast cancer in humans. In this study, the physical swelling method was used to prepare halofuginone-loaded D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) polymer micelles nano-thermosensitive hydrogels (HTPM-gel). Furthermore, HTPM-gel was investigated via characterization, morphology, properties such as swelling experiment and in vitro release with reflecting its splendid nature. Moreover, HTPM-gel was further examined its capability to anti-proliferation, anti-migration, and anti-invasion. Ultimately, HTPM-gel was investigated for its in vivo anticancer activity in the post-operative metastatic and angiogenic canine mammary carcinoma. HTPM-gel presented spherical under transmission electron microscope (TEM) and represented grid structure under scanning electron microscope (SEM), with hydrodynamic diameter (HD) of 20.25 ± 2.5 nm and zeta potential (ZP) of 15.10 ± 1.82 mV. Additionally, HTPM-gel own excellent properties comprised of pH-dependent swelling behavior, sustained release behavior. To impede the migration, invasion, and proliferation of CMT-U27 cells, we tested the efficacy of HTPM-gel. Evaluation of in vivo anti-tumor efficacy demonstrates HTPM-gel exhibit a splendid anti-metastasis and anti-angiogenic ability, with exhibiting ideal biocompatibility. Notably, HTPM-gel also inhibited the scar formation in the healing process after surgery. In summary, HTPM-gel exhibited anti-metastasis and anti-angiogenic and scar repair features. According to the results of this study, HTPM-gel has encouraging clinical potential to treat tumors with multifunctional hydrogel., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

The manipulation of T cell metabolism to enhance anti-tumor activity is an area of active investigation. Here, we report that activating the amino acid starvation response in effector CD8 + T cells ex vivo using the general control non-depressible 2 (GCN2) agonist halofuginone (halo) enhances oxidative metabolism and effector function. Mechanistically, we identified autophagy coupled with the CD98-mTOR axis as key downstream mediators of the phenotype induced by halo treatment. The adoptive transfer of halo-treated CD8 + T cells into tumor-bearing mice led to robust tumor control and curative responses. Halo-treated T cells synergized in vivo with a 4-1BB agonistic antibody to control tumor growth in a mouse model resistant to immunotherapy. Importantly, treatment of human CD8 + T cells with halo resulted in similar metabolic and functional reprogramming. These findings demonstrate that activating the amino acid starvation response with the GCN2 agonist halo can enhance T cell metabolism and anti-tumor activity., Competing Interests: Declaration of interests P.S.O. is on the SAB for Providence Therapeutics, Treadwell Therapeutics, and Egle Therapeutics and holds SRA with EMD Serono. The authors have filed a patent pertaining to the use of halo to enhance immunotherapy., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Halofuginone is a potential anti-malarial drug, which could exist as three possible tautomers. Herein, using density functional theory (DFT), and handling the solvent effects with the PCM model, the tautomerism of halofuginone was investigated. Intramolecular H-bonds play an important role in the stability of the tautomers. The conformer H1a is the most stable. Noncovalent interactions of the H1a conformer with the armchair (5,5) single-wall carbon nanotubes and γ-Fe2O3 nanoparticles were explored in several manners. The most stable form of them was determined. The intermolecular H-bonds play a substantial role in the energy behavior of the interaction between γ-Fe2O3 nanoparticles and halofuginone.

Malignant mesothelioma is a rare but aggressive form of malignancy, which is difficult to diagnose and is resistant to current chemotherapeutic treatment options. Molecular techniques have been used to investigate the mechanisms of action and the beneficial therapeutic effects of halofuginone (HF) in several cancers but not malignant mesotheliomas. In this study, the antiproliferative and apoptotic effects of HF were investigated through its ability to deregulate EGFR downstream signalling cascade proteins in the pathologically aggressive malignant mesothelioma and non‐small‐cell lung cancer cells. We showed that administration of HF at nanomolar concentrations induced a dose‐dependent reduction in the viability of cancer cells, made cell cycle arrest, inhibited proliferation of cancer cells via STAT3 and ERK1/2 pathways and triggered the apoptotic cascade via p38MAPK. We demonstrated that the apoptotic cell death mechanism was mediated by enhanced activation of caspase‐3 and concomitant PARP cleavage, downregulation of Bcl‐2 and upregulation of Bax in both malignant mesothelioma and lung cancer cells. In particular, we demonstrated that cancer cells were more sensitive to HF treatment than normal mesothelial cells. Taken together, this study suggests that HF exerts its anticancer effects in lung‐derived cancers by targeting signal transduction pathways mainly through deregulation of ERK1/2, STAT3 and p38MAPK to reduce cancer cell viability, induce cell cycle arrest and apoptotic cell death. Thus, HF might be considered as a potential agent against malignant mesothelioma and/or lung cancer cells. [ABSTRACT FROM AUTHOR]

• Dysferlinopathies are a group of late-onset muscular dystrophies. • Impaired fusion of primary dysf −/− myoblasts from young mice. • Elevated fibrotic symptoms are found in dysf−/− mice at 4 weeks of age. • Early halofuginone treatment rescues dysf−/− mouse muscle histopathology. • Early halofuginone treatment improves myoblast fusion in dysf−/− mice. Dysferlinopathies are a non-lethal group of late-onset muscular dystrophies. Here, we evaluated the fusion ability of primary myoblasts from young dysf−/− mice and the muscle histopathology prior to, and during early stages of disease onset. The ability of primary myoblasts of 5-week-old dysf−/− mice to form large myotubes was delayed compared to their wild-type counterparts, as evaluated by scanning electron microscopy. However, their fusion activity, as reflected by the presence of actin filaments connecting several cells, was enhanced by the antifibrotic drug halofuginone. Early dystrophic signs were already apparent in 4-week-old dysf−/− mice; their collagen level was double that in wild-type mice and continued to rise until 5 months of age. Continuous treatment with halofuginone from 4 weeks to 5 months of age reduced muscle fibrosis in a phosphorylated-Smad3 inhibition-related manner. Halofuginone also enhanced myofiber hypertrophy, reduced the percentage of centrally nucleated myofibers, and increased muscle performance. Together, the data suggest an inhibitory effect of halofuginone on the muscle histopathology at very early stages of dysferlinopathy, and enhancement of muscle performance. These results offer new opportunities for early pharmaceutical treatment in dysferlinopathies with favorable outcomes at later stages of life. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) is characterized by neutrophilic infiltration, uncontrolled oxidative stress and inflammatory processes. Despite various therapeutic regimes having been performed, there remains no effective pharmacotherapy available to treat ALI. Halofuginone (HF), a ketone isolated from Dichroa febrifuga, exhibits significant anti-inflammatory and antifibrotic effects. Dexamethasone (DEX), a synthetic glucocorticoid, has been routinely used as an adjuvant therapy in treating inflammatory diseases, including ALI. The present study aimed to investigate the effects of the combination of HF and DEX in the treatment of ALI. The present results suggested that the simultaneous administration of HF and DEX markedly decreased the level of pro-inflammatory cytokines and increased the level of anti-inflammatory cytokines, as assessed by western blot analysis. In addition, HF and DEX effectively decreased nuclear factor-κB activity via suppressing the phosphorylation of P65 in lipopolysaccharide (LPS)-induced human pulmonary alveolar epithelial cells (HPAEpiC) and lung tissues extracted from ALI rats, as determined by immunofluorescence. Furthermore, in vivo experiments demonstrated that the combination of HF and DEX in LPS-induced ALI rats defended against lung fibrosis, perivascular inflammation, congestion and edema of pulmonary alveoli, as assessed by histopathological analysis, TUNEL staining and immunohistochemistry assay. Taken together, the present study indicated the synergistic effect of HF and DEX on LPS-induced ALI in HPAEpiC cells and a rat model. These results offer a novel therapeutic approach for the treatment of ALI. [ABSTRACT FROM AUTHOR]

Halofuginone (HF) is an extract from the widely used traditional Chinese medicine (TCM) Dichroa febrifuga that facilitates the recovery of wounds and attenuates hepatic fibrosis. However, the role of HF in the epithelial-mesenchymal transition (EMT) of IPEC-J2 cells remains unclear. The current study explored the anti-EMT effect of HF in IPEC-J2 cells and illustrates its molecular mechanism. Transforming growth factor β1 (TGF-β1), as a recognized profibrogenic cytokine, decreased the level of the epithelial marker E-cadherin and increased the level of the mesenchymal markers, such as N-cadherin, fibronectin (FN), vimentin (Vim), and α-smooth muscle actin (α-SMA), in IPEC-J2 cells depending on the exposure time and dose. HF markedly prevented the EMT induced by TGF-β1. Dissection of the mechanism revealed that HF inhibited IPEC-J2 cell EMT via modulating the phosphorylation of SMAD2/3 and the SMAD2/3-SMAD4 complex nuclear translocation. Furthermore, HF could promote the phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which modulates the SMAD signaling pathway. These results suggested that HF inhibits TGF-β1-induced EMT in IPEC-J2 cells through the eIF2α/SMAD signaling pathway. Our findings suggest that HF can serve as a potential anti-EMT agent in intestinal fibrosis therapy. [ABSTRACT FROM AUTHOR]

Background: Halofuginone is an alkaloid febrifugine analogue and bioactive molecule that was isolated incidentally from the Dichroa febrifuga plant. The therapeutic efficacy of halofuginone in parasitic infections, scleroderma, inflammation, and fibrosis-related diseases, as well as in some types of cancer, has been previously reported. The effects of halofuginone on nasal mucosal damage are not yet known. Objective: The aim of this study was to investigate the potential effect of topically applied halofuginone on wound healing in the mechanically injured nasal mucosa of rats. Methods: A unilateral mucosal wound was created in the nasal cavity of 32 rats (aged 4 weeks) using the brushing technique. These rats were randomly divided into 4 groups. Although the control group did not receive an intervention, a dry pad, a saline-impregnated pad, or a pad impregnated with halofuginone were placed in the rats of the other 3 groups and left for 5 minutes. Rats were sacrificed on the 14th day, and a histological examination was performed. The nasal mucosa was assessed via hematoxylin-eosin and Masson's trichrome staining. Results: There were no statistically significant differences in epithelial thickness, inflammation, goblet cell formation, and epithelial disarray values between the halofuginone group and the control group (P >.05). The subepithelial thickness was significantly decreased in the saline-treated group and the halofuginone-treated group (P <.05), but a significantly lower level of subepithelial fibrosis was only observed in the halofuginone group compared to the other groups (P <.05). Conclusions: Topical halofuginone administration reduces the development of fibrosis and subepithelial edema after experimentally induced nasal mucosal injury, but it does not exert therapeutic or preventive effects on epithelial damage, inflammation, and goblet cell hyperplasia. [ABSTRACT FROM AUTHOR]