Rapamycin Derivatives Research Articles

Temsirolimus is a promising immunomodulatory agent for enhanced transplantation outcomes

BackgroundIdentifying effective immunosuppressive strategies is critical for addressing immunological rejection following organ transplantation. This study explores the potential immunosuppressive effects and mechanisms of temsirolimus, a rapamycin derivative, in organ transplantation. MethodsA mouse cardiac allograft model was established using a cervical cannula technique with BALB/c donors and C57BL/6 recipients. Mice were administered temsirolimus intragastrically and graft survival was evaluated. Histological staining was used to assess pathological changes. The BrdU assay was used to measure splenic T cell proliferation. Flow cytometry was used to quantify regulatory T cells (Tregs), CD4+ T cells, and CD8+ T cells. ELISA and qPCR assays were used to determine Foxp3, IL-4, IFN-γ, and TGF-β expression. ResultsTemsirolimus displayed potent immunosuppressive effects at 20 mg/kg/day, significantly inhibiting T cell proliferation (84.6%, P < 0.0001) and prolonging graft survival (median 49 days vs. 8.5 days in controls, P < 0.0001). However, median survival decreased to 34.5 days upon withdrawal. Temsirolimus also reduced splenic CD4+ and CD8+ T cells (2.85% and 2.92%, P < 0.001) and antibody levels (IgM, IgG1, IgG2) by 11.85–29.09% (P < 0.0001) and increased Tregs, Foxp3, IL-4 (P < 0.01), and TGF-β (P < 0.05), while decreasing IFN-γ (P < 0.001). ConclusionsTemsirolimus exhibited potent immunosuppressive effects, emerging as a strong candidate to mitigate organ transplant rejection.

Cyclosporines Antagonize the Antiviral Activity of IFITMProteins by Redistributing Them toward the Golgi Apparatus.

Interferon-induced transmembrane proteins (IFITMs) block the fusion of diverse enveloped viruses, likely through increasing the cell membrane's rigidity. Previous studies have reported that the antiviral activity of the IFITM family member, IFITM3, is antagonized by cell pretreatment with rapamycin derivatives and cyclosporines A and H (CsA and CsH) that promote the degradation of IFITM3. Here, we show that CsA and CsH potently enhance virus fusion with IFITM1- and IFITM3-expressing cells by inducing their rapid relocalization from the plasma membrane and endosomes, respectively, towards the Golgi. This relocalization is not associated with a significant degradation of IFITMs. Although prolonged exposure to CsA induces IFITM3 degradation in cells expressing low endogenous levels of this protein, its levels remain largely unchanged in interferon-treated cells or cells ectopically expressing IFITM3. Importantly, the CsA-mediated redistribution of IFITMs to the Golgi occurs on a much shorter time scale than degradation and thus likely represents the primary mechanism of enhancement of virus entry. We further show that rapamycin also induces IFITM relocalization toward the Golgi, albeit less efficiently than cyclosporines. Our findings highlight the importance of regulation of IFITM trafficking for its antiviral activity and reveal a novel mechanism of the cyclosporine-mediated modulation of cell susceptibility to enveloped virus infection.

Advances in the mTOR signaling pathway and its inhibitor rapamycin in epilepsy.

Epilepsy is one of the most common and serious brain syndromes and has adverse consequences on a patient's neurobiological, cognitive, psychological, and social wellbeing, thereby threatening their quality of life. Some patients with epilepsy experiencepoor treatment effects due to the unclear pathophysiological mechanisms of the syndrome. Dysregulation of the mammalian target of the rapamycin (mTOR) pathway is thought to play an important role in the onset and progression of some epilepsies. This review summarizes the role of the mTOR signaling pathway in the pathogenesis of epilepsy and the prospects for the use of mTOR inhibitors. The mTOR pathway functions as a vital mediator in epilepsy development through diverse mechanisms, indicating that the it has great potential as an effective target for epilepsy therapy. The excessive activation of mTOR signaling pathway leads to structural changes in neurons, inhibits autophagy, exacerbates neuron damage, affects mossy fiber sprouting, enhances neuronal excitability, increases neuroinflammation, and is closely associated with tau upregulation in epilepsy. A growing number of studies have demonstrated that mTOR inhibitors exhibit significant antiepileptic effects in both clinical applications and animal models. Specifically, rapamycin, a specific inhibitor of TOR, reduces the intensity and frequency of seizures. Clinical studies in patients with tuberous sclerosis complex have shown that rapamycin has the function of reducing seizures and improving this disease. Everolimus, a chemically modified derivative of rapamycin, has been approved as an added treatment to other antiepileptic medicines. Further explorations are needed to evaluate the therapeutic efficacy and application value of mTOR inhibitors in epilepsy. Targeting the mTOR signaling pathway provides a promising prospect for the treatment of epilepsy.

Molecular and therapeutic insights of rapamycin: a multi-faceted drug from Streptomyces hygroscopicus.

The advancement in pharmaceutical research has led to the discovery and development of new combinatorial life-saving drugs. Rapamycin is a macrolide compound produced from Streptomyces hygroscopicus. Rapamycin and its derivatives are one of the promising sources of drug with broad spectrum applications in the medical field. In recent times, rapamycin has gained significant attention as of its activity against cytokine storm in COVID-19 patients. Rapamycin and its derivatives have more potency when compared to other prevailing drugs. Initially, it has been used exclusively as an anti-fungal drug. Currently rapamycin has been widely used as an immunosuppressant. Rapamycin is a multifaceted drug; it has anti-cancer, anti-viral and anti-aging potentials. Rapamycin has its specific action on mTOR signaling pathway. mTOR has been identified as a key regulator of different pathways. There will be an increased demand for rapamycin, because it has lesser adverse effects when compared to steroids. Currently researchers are focused on the production of effective rapamycin derivatives to combat the growing demand of this wonder drug. The main focus of the current review is to explore the origin, development, molecular mechanistic action, and the current therapeutic aspects of rapamycin. Also, this review article revealed the potential of rapamycin and the progress of rapamycin research. This helps in understanding the exact potency of the drug and could facilitate further studies that could fill in the existing knowledge gaps. The study also gathers significant data pertaining to the gene clusters and biosynthetic pathways involved in the synthesis and production of this multi-faceted drug. In addition, an insight into the mechanism of action of the drug and important derivatives of rapamycin has been expounded. The fillings of the current review, aids in understanding the underlying molecular mechanism, strain improvement, optimization and production of rapamycin derivatives.

Developmental and reproduction toxicity studies of Biolimus A9 in SD rats

Biolimus A9 (BA9) is a novel rapamycin derivative. In this report we evaluated the potential toxicity of BA9 in a developmental and reproduction toxicity study (segment Ⅰ, Ⅱ, Ⅲ). In segment I, body weight gains in F0 rats receiving 0.80 mg/kg/day were decreased. A lower fertility index of males was observed and females failed to become pregnant in the 0.80 mg/kg/day group. The number of live fetuses and implantations were decreased while the number of dead fetuses, resorptions, and implantation losses were increased in the 0.12 mg/kg/day group. In segment Ⅱ, maternal toxicity: body weight gains in F0 females receiving 0.036 and 0.090 mg/kg/day group were decreased. Embryo toxicity: In the 0.090 mg/kg/day group, weights and body lengths of fetuses were decreased, the numbers of viable fetuses was decreased and resorbed fetuses increased. Teratogenic effects: The percent of visceral variations and skeletal variations were both increased in the 0.090 mg/kg/day group. In segment Ⅲ, dosing F0 rats with BA9 at dose levels of 0.12 and 0.80 mg/kg/day resulted in reproductive and maternal toxicity, consisting of prolonged labor, dystocia, increased mortality, along with reductions in lactation food consumption. F1 rats in the 0.12 mg/kg/day group showed reproductive and developmental toxicity consisting of body weight decreases, decreased food consumption after weaning and a reduction in the gestation index of pregnant rats. Based on these findings, the no-observed-adverse-effect-level (NOAEL) of BA9 toxicity in segment Ⅰ and Ⅲ was 0.02 mg/kg/day. The NOAEL in segment Ⅱ was 0.015 mg/kg/day.

Optimization of the Everolimus Intermediate Preparation Process: Impurity Identification Followed by One-Pot Transformation to the Desired Product

An investigation of the everolimus intermediate PG-EVR preparation process resulted in the identification of three impurities PG-D,E,F constantly formed as byproducts in large amounts. Their structures were reliably established based on two-dimensional (2D) NMR and liquid chromatography–high-resolution tandem mass spectrometry (LC–HRMS/MS) data as dialkylated rapamycin derivatives. Finding that one of these impurities PG-E is hydrolyzable to the target everolimus intermediate resulted in optimized technology based on changing the selectivity of byproduct formation to PG-E as the major one followed by its one-pot quantitative transformation to PG-EVR. The yield of the everolimus intermediate increased by 10% under mild conditions, while lower amounts of reagents and solvents were required. The active pharmaceutical ingredient (API) that meets the requirements of the European Pharmacopoeia was prepared from thus obtained intermediate with no need for high-performance liquid chromatography (HPLC) purification, indicating a significant increase in the efficiency of large-scale production using new technology.

Anti-Cancer Effects of Zotarolimus Combined with 5-Fluorouracil Treatment in HCT-116 Colorectal Cancer-Bearing BALB/c Nude Mice.

Zotarolimus is a semi-synthetic derivative of rapamycin and an inhibitor of mammalian target of rapamycin (mTOR) signaling. Currently, zotarolimus is used to prolong the survival time of organ grafts, but it is also a novel immunosuppressive agent with potent anti-proliferative activity. Here, we examine the anti-tumor effect of zotarolimus, alone and in combination with 5-fluorouracil, on HCT-116 colorectal adenocarcinoma cells implanted in BALB/c nude mice. Compared with the control mice, mice treated with zotarolimus or zotarolimus combined with 5-FU showed retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; reduced inflammation-related factors such as IL-1β, TNF-α, and cyclooxygenase-2 (COX-2) protein; and inhibited metastasis-related factors such as CD44, epidermal growth factor receptor (EGFR), transforming growth factor β (TGF-β), and vascular endothelial growth factor (VEGF). Notably, mice treated with a combination of zotarolimus and 5-FU showed significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with mice treated with 5-FU or zotarolimus alone, indicating a strong synergistic effect. This in vivo study confirms that zotarolimus or zotarolimus combined with 5-FU can be used to retard colorectal adenocarcinoma growth and inhibit tumorigenesis. Our results suggest that zotarolimus may increase the chemo-sensitization of tumor cells. Therefore, zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents in the treatment of human colon adenocarcinoma. Future research on zotarolimus may lead to the development of new therapeutic strategies.

Hemiacetal-less rapamycin derivatives designed and produced by genetic engineering of a type I polyketide synthase

Engineering polyketide synthases is one of the most promising ways of producing a variety of polyketide derivatives. Exploring the undiscovered chemical space of this medicinally important class of middle molecular weight natural products will aid in the development of improved drugs in the future. In previous work, we established methodology designated ‘module editing’ to precisely manipulate polyketide synthase genes cloned in a bacterial artificial chromosome. Here, in the course of investigating the engineering capacity of the rapamycin PKS, novel rapamycin derivatives 1–4, which lack the hemiacetal moiety, were produced through the heterologous expression of engineered variants of the rapamycin PKS. Three kinds of module deletions in the polyketide synthase RapC were designed, and the genetically engineered vectors were prepared by the in vitro module editing technique. Streptomyces avermitilis SUKA34 transformed with these edited PKSs produced new rapamycin derivatives. The planar structures of 1–4 established based on 1D and 2D NMR, ESI–TOF–MS and UV spectra revealed that 2 and 3 had skeletons well-matched to the designs, but 1 and 4 did not. The observations provide important insights into the mechanisms of the later steps of rapamycin skeletal formation as well as the ketone-forming oxygenase RapJ.

The Anti-Cancer Effects of a Zotarolimus and 5-Fluorouracil Combination Treatment on A549 Cell-Derived Tumors in BALB/c Nude Mice.

Zotarolimus is a semi-synthetic derivative of rapamycin and a novel immunosuppressive agent used to prevent graft rejection. The pharmacological pathway of zotarolimus restricts the kinase activity of the mammalian target of rapamycin (mTOR), which potentially leads to reductions in cell division, cell growth, cell proliferation, and inflammation. These pathways have a critical influence on tumorigenesis. This study aims to examine the anti-tumor effect of zotarolimus or zotarolimus combined with 5-fluorouracil (5-FU) on A549 human lung adenocarcinoma cell line implanted in BALB/c nude mice by estimating tumor growth, apoptosis expression, inflammation, and metastasis. We established A549 xenografts in nude mice, following which we randomly divided the mice into four groups: control, 5-FU (100 mg/kg/week), zotarolimus (2 mg/kg/day), and zotarolimus combined with 5-FU. Compared the results with those for control mice, we found that mice treated with zotarolimus or zotarolimus combined with 5-FU retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; decreased inflammation cytokines levels (e.g., IL-1β, TNF-α, and IL-6); reduced inflammation-related factors such as cyclooxygenase-2 (COX-2) protein and nuclear factor-κB (NF-κB) mRNA; enhanced anti-inflammation-related factors including IL-10 and inhibitor of NF-κB kinase α (IκBα) mRNA; and inhibited metastasis-related factors such as transforming growth factor β (TGF-β), CD44, epidermal growth factor receptor (EGFR), and vascular endothelial growth factor (VEGF). Notably, mice treated with zotarolimus combined with 5-FU had significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with the groups of mice treated with 5-FU or zotarolimus alone. The in vivo study confirmed that zotarolimus or zotarolimus combined with 5-FU could retard lung adenocarcinoma growth and inhibit tumorigenesis. Zotarolimus and 5-FU were found to have an obvious synergistic tumor-inhibiting effect on lung adenocarcinoma. Therefore, both zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents for treatment of human lung adenocarcinoma.

The Putative Role of mTOR Inhibitors in Non-tuberous Sclerosis Complex-Related Epilepsy.

Epilepsy affects ~5 out of every 10,000 children per year. Up to one-third of these children have medically refractory epilepsy, with limited to no options for improved seizure control. mTOR, a ubiquitous 289 kDa serine/threonine kinase in the phosphatidylinositol 3-kinase (PI3K)-related kinases (PIKK) family, is dysregulated in a number of human diseases, including tuberous sclerosis complex (TSC) and epilepsy. In cell models of epilepsy and TSC, rapamycin, an mTOR inhibitor, has been shown to decrease seizure frequency and duration, and positively affect cell growth and morphology. Rapamycin has also been shown to prevent or improve epilepsy and prolong survival in animal models of TSC. To date, clinical studies looking at the effects of mTOR inhibitors on the reduction of seizures have mainly focused on patients with TSC. Everolimus (Novartis Pharmaceuticals), a chemically modified rapamycin derivative, has been shown to reduce seizure frequency with reasonable safety and tolerability. Mutations in mTOR or the mTOR pathway have been found in hemimegalencephaly (HME) and focal cortical dysplasias (FCDs), both of which are highly correlated with medically refractory epilepsy. Given the evidence to date, a logical next step is to investigate the role of mTOR inhibitors in the treatment of children with medically refractory non-TSC epilepsy, particularly those children who have also failed resective surgery.

Novel brain permeant mTORC1/2 inhibitors are as efficacious as rapamycin or everolimus in mouse models of acquired partial epilepsy and tuberous sclerosis complex

Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative (“rapalog”) everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of two novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors, PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1GFAP CKO mice, a well-characterized mouse model of epilepsy in TSC. During prolonged treatment of IHK mice with rapamycin, everolimus, PQR620, PQR626, or PQR530; only PQR620 exerted a transient antiseizure effect on SRS, at well tolerated doses whereas the other compounds were ineffective. In contrast, all of the examined compounds markedly suppressed SRS in Tsc1GFAP CKO mice during chronic treatment at well tolerated doses. Thus, against our expectation, no clear differences in antiseizure efficacy were found across the three classes of mTOR inhibitors examined in mouse models of genetic and acquired epilepsies. The main advantage of the novel 1,3,5-triazine derivatives is their excellent tolerability compared to rapalogs, which would favor their development as new therapies for TORopathies such as TSC.

Structural insights into CYP107G1 from rapamycin-producing Streptomyces rapamycinicus

Rapamycin is a clinically important macrolide agent with immunosuppressant and antiproliferative properties, produced by the actinobacterium, Streptomyces rapamycinicus. Two cytochrome P450 enzymes are involved in the biosynthesis of rapamycin. CYP107G1 and CYP122A2 catalyze the oxidation reactions of C27 and C9 of pre-rapamycin, respectively. To understand the structural and biochemical features of P450 enzymes in rapamycin biosynthesis, the CYP107G1 and CYP122A2 genes were cloned, their recombinant proteins were expressed in Escherichia coli, and the purified enzymes were characterized. Both enzymes displayed low spin states in the absolute spectra of ferric forms, and the titrations with rapamycin induced type I spectral changes with Kd values of 4.4 ± 0.4 and 3.0 ± 0.3 μM for CYP107G1 and CYP122A2, respectively. The X-ray crystal structures of CYP107G1 and its co-crystal complex with everolimus, a clinical rapamycin derivative, were determined at resolutions of 2.9 and 3.0 Å, respectively. The overall structure of CYP107G1 adopts the canonical scaffold of cytochrome P450 and possesses large substrate pocket. The distal face of the heme group is exposed to solvents to accommodate macrolide access. When the structure of the everolimus-bound CYP107G1 complex (CYP107G1-Eve) was compared to that of the ligand-free CYP107G1 form, no significant conformational change was observed. Hence, CYP107G1 has a relatively rigid structure with versatile loops to accommodate a bulky substrate. The everolimus molecule is bound to the substrate-binding pocket in the shape of a squeezed donut, and its elongated structure is bound perpendicular to a planar heme plane and I-helix.

Potential new therapy of Rapalink-1, a new generation mammalian target of rapamycin inhibitor, against sunitinib-resistant renal cell carcinoma.

Sunitinib, a multitargeted receptor tyrosine kinase inhibitor including vascular endothelial growth factor, has been widely used as a first‐line treatment against metastatic renal cell carcinoma (mRCC). However, mRCC often acquires resistance to sunitinib, rendering it difficult to treat with this agent. Recently, Rapalink‐1, a drug that links rapamycin and the mTOR kinase inhibitor MLN0128, has been developed with excellent therapeutic effects against breast cancer cells carrying mTOR resistance mutations. The aim of the present study was to evaluate the in vitro and in vivo therapeutic efficacy of Rapalink‐1 against renal cell carcinoma (RCC) compared to temsirolimus, which is commonly used as a small molecule inhibitor of mTOR and is a derivative of rapamycin. In comparison with temsirolimus, Rapalink‐1 showed significantly greater effects against proliferation, migration, invasion and cFolony formation in sunitinib‐naïve RCC cells. Inhibition was achieved through suppression of the phosphorylation of substrates in the mTOR signal pathway, such as p70S6K, eukaryotic translation initiation factor 4E‐binding protein 1 (4EBP1) and AKT. In addition, Rapalink‐1 had greater tumor suppressive effects than temsirolimus against the sunitinib‐resistant 786‐o cell line (SU‐R 786‐o), which we had previously established, as well as 3 additional SU‐R cell lines established here. RNA sequencing showed that Rapalink‐1 suppressed not only the mTOR signaling pathway but also a part of the MAPK signaling pathway, the ErbB signaling pathway and ABC transporters that were associated with resistance to several drugs. Our study suggests the possibility of a new treatment option for patients with RCC that is either sunitinib‐sensitive or sunitinib‐resistant.

Autophagy and mTOR signaling during intervertebral disc aging and degeneration.

Degenerative disc disease is a highly prevalent, global health problem that represents the primary cause of back pain and is associated with neurological disorders, including radiculopathy, myelopathy, and paralysis, resulting in worker disability and socioeconomic burdens. The intervertebral disc is the largest avascular organ in the body, and degeneration is suspected to be linked to nutritional deficiencies. Autophagy, the process through which cells self‐digest and recycle damaged components, is an important cell survival mechanism under stress conditions, especially nutrient deprivation. Autophagy is negatively controlled by the mammalian target of rapamycin (mTOR) signaling pathway. mTOR is a serine/threonine kinase that detects nutrient availability to trigger the activation of cell growth and protein synthesis pathways. Thus, resident disc cells may utilize autophagy and mTOR signaling to cope with harsh low‐nutrient conditions, such as low glucose, low oxygen, and low pH. We performed rabbit and human disc cell and tissue studies to elucidate the involvement and roles played by autophagy and mTOR signaling in the intervertebral disc. In vitro serum and nutrient deprivation studies resulted in decreased disc cell proliferation and metabolic activity and increased apoptosis and senescence, in addition to increased autophagy. The selective RNA interference‐mediated and pharmacological inhibition of mTOR complex 1 (mTORC1) was protective against inflammation‐induced disc cellular apoptosis, senescence, and extracellular matrix catabolism, through the induction of autophagy and the activation of the Akt‐signaling network. Although temsirolimus, a rapamycin derivative with improved water solubility, was the most effective mTORC1 inhibitor tested, dual mTOR inhibitors, capable of blocking multiple mTOR complexes, did not rescue disc cells. In vivo, high levels of mTOR‐signaling molecule expression and phosphorylation were observed in human intermediately degenerated discs and decreased with age. A mechanistic understanding of autophagy and mTOR signaling can provide a basis for the development of biological therapies to treat degenerative disc disease.

A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor.

The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure-activity relationship (SAR) studies led to the identification of compound 12b with a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.

Next Generation Strategies for Geroprotection via mTORC1 Inhibition

Inhibition of mTORC1 (mechanistic Target Of Rapamycin Complex 1) with the pharmaceutical rapamycin prolongs the lifespan and healthspan of model organisms including rodents, with evidence now emerging that rapamycin and its analogs may also have rejuvenative effects in dogs and humans. However, the side effects associated with long-term rapamycin treatment, many of which are due to inhibition of a second mTOR complex, mTORC2, have seemed to preclude the routine use of rapamycin as a therapy for age-related diseases. Here, we discuss recent findings suggesting that strong, chronic inhibition of both mTOR complexes may not be necessary to realize the geroprotective effects of rapamycin. Instead, modestly but specifically inhibiting mTORC1 via a variety of emerging techniques, including intermittent or transient treatment with rapamycin derivatives, or specific dietary regimens, may be sufficient to promote health and longevity with reduced side effects. We will also discuss prospects for the development of new molecules that, by harnessing the detailed molecular understanding of mTORC1 signaling developed over the last decade, will provide new routes to the selective inhibition of mTORC1. We conclude that therapies based on the selective inhibition of mTORC1 may soon permit the safer treatment of diseases of aging.

A rapamycin derivative, biolimus, preferentially activates autophagy in vascular smooth muscle cells

Although rapamycin is a well-known conformational inhibitor of mTORC1, it is now widely used for treating arterial restenosis. Various rapamycin analogues (rapalogue) have been made for applying to drug-eluting stents. Here we show that two major rapalogues, everolimus and biolimus, exert a differential effect on the mTORC1-mediated signaling pathways in vascular smooth muscle cells. In balloon-injured carotid arteries, both rapalogues strongly inhibit neointimal hyperplasia. Signaling pathway analyses reveal that everolimus exert cytotoxicity by increasing cellular reactive oxygen species and consequently reduce energy metabolism. By contrast, biolimus confers a preferential induction of autophagy by more strongly activating major autophagy regulator, ULK1, in vascular smooth muscle cells than everolimus does. As a consequence, the implantation of biolimus-eluting stent reduces endothelial loss, which in turn reduces inflammation, in porcine coronary arteries. Thus, this study reveals that a chemical derivatization can cause a change among mTORC1-dependent signaling pathways in vascular smooth muscle cells, thereby enabling to elicit a differential efficacy on arterial restenosis.

Kaposi's Sarcoma-Associated Herpesvirus Latency Locus Renders B Cells Hyperresponsive to Secondary Infections.

Kaposi's sarcoma-associated herpesvirus (KSHV) induces B cell hyperplasia and neoplasia, such as multicentric Castleman's disease (MCD) and primary effusion lymphoma (PEL). To explore KSHV-induced B cell reprogramming in vivo, we expressed the KSHV latency locus, inclusive of all viral microRNAs (miRNAs), in B cells of transgenic mice in the absence of the inhibitory FcγRIIB receptor. The BALB/c strain was chosen as this is the preferred model to study B cell differentiation. The mice developed hyperglobulinemia, plasmacytosis, and B lymphoid hyperplasia. This phenotype was ameliorated by everolimus, which is a rapamycin derivative used for the treatment of mantle cell lymphoma. KSHV latency mice exhibited hyperresponsiveness to the T-dependent (TD) antigen mimic anti-CD40 and increased incidence of pristane-induced inflammation. Lastly, the adaptive immunity against a secondary infection with Zika virus (ZIKV) was markedly enhanced. These phenotypes are consistent with KSHV lowering the activation threshold of latently infected B cells, which may be beneficial in areas of endemicity, where KSHV is acquired in childhood and infections are common.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latency in B cells and is stringently linked to primary effusion lymphoma (PEL) and the premalignant B cell hyperplasia multicentric Castleman's disease (MCD). To investigate potential genetic background effects, we expressed the KSHV miRNAs in BALB/c transgenic mice. BALB/c mice are the preferred strain for B cell hybridoma development because of their propensity to develop predictable B cell responses to antigen. The BALB/c latency mice exhibited a higher incidence of B cell hyperplasia as well as sustained hyperglobulinemia. The development of neutralizing antibodies against ZIKV was augmented in BALB/c latency mice. Hyperglobulinemia was dampened by everolimus, a derivative of rapamycin, suggesting a role for mTOR inhibitors in managing immune activation, which is hallmark of KSHV infection as well as HIV infection.

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

The benefit of everolimus in recurrent/epithelioid angiosarcoma patients: Case reports and literature review.

Epithelioid angiosarcoma (EA) is a kind of rare malignant soft tissue sarcoma, with high recurrence/metastatic rate and poor prognosis. To date, no effective standardized treatment regimen has been available for patients with recurrence/metastatic EA. Everolimus is an oral rapamycin derivative that highly inhibits the mechanistic target of rapamycin(mTOR) signal pathway. Previous studies have suggested that everolimus is effective and safe in some soft tissue sarcoma. We reported two cases with recurrence/metastatic EA, who received everolimus after failure of surgery, radiotherapy, chemotherapy or interventional therapy. Two cases obtained clinical benefit within 1 week, and were evaluated as partial response (PR). The progression free survival (PFS) time was nearly 12.0 and 6.0 months, respectively. The overall survival (OS) time was 18.0 and 10.0 months, respectively. The main adverse event was stomatitis syndrome (grade 1-2), which was well controllable and tolerable. It indicated that everolimus may be more beneficial for recurrence/metastatic EA patients.