Discovery of a long half-life AURKA inhibitor to treat MYC-amplified solid tumors as a monotherapy and in combination with everolimus.

Aurora kinase inhibitors such as alisertib can destabilize MYC-family oncoproteins and have demonstrated compelling anti-tumor efficacy. In this study, we report 6K465, a novel pyrimidine-based Aurora A (AURKA) inhibitor that reduces levels of c-MYC and N-MYC oncoproteins more potently than alisertib. In an analysis of the antiproliferative effect of 6K465, the sensitivities of small cell lung cancer (SCLC) and breast cancer (BC) cell lines to 6K465 were strongly associated with the protein levels of c-MYC and/or N-MYC. We also report DBPR728, an acyl-based prodrug of 6K465 bearing fewer hydrogen-bond donors that exhibited 10-fold improved oral bioavailability. DBPR728 induced durable tumor regression of c-MYC- and/or N-MYC- overexpressing xenografts including SCLC, triple-negative breast cancer (TNBC), hepatocellular carcinoma and medulloblastoma using a 5-on-2-off or once-a-week dosing regimen on a 21-day cycle. A single oral dose of DBPR728 at 300 mg/kg induced c-MYC reduction and cell apoptosis in the tumor xenografts for more than 7 days. The inhibitory effect of DBPR728 at a reduced dosing frequency was attributed to its uniquely high tumor/plasma ratio (3.6-fold within 7 days) and the long tumor half-life of active moiety 6K465. Furthermore, DBPR728 was found to synergize with the mTOR inhibitor everolimus to suppress c-MYC- or N-MYC- driven SCLC. Collectively, these results suggest DBPR728 has the potential to treat cancers overexpressing c-MYC- and/or N-MYC.

Analysis of structure-activity relationship of indol-3-yl-N-phenylcarbamic amides as potent STING inhibitors.

A structure-activity relationship (SAR) study of stimulator of interferon gene (STING) inhibition was performed using a series of indol-3-yl-N-phenylcarbamic amides and indol-2-yl-N-phenylcarbamic amides. Among these analogs, compounds 10, 13, 15, 19, and 21 inhibited the phosphorylation of STING and interferon regulatory factor 3 (IRF3) to a greater extent than the reference compound, H-151. All five analogs showed stronger STING inhibition than H-151 on the 2',3'-cyclic GMP-AMP-induced expression of interferon regulatory factors (IRFs) in a STINGR232 knock-in THP-1 reporter cell line. The half-maximal inhibitory concentration of the most potent compound, 21, was 11.5 nM. The molecular docking analysis of compound 21 and STING combined with the SAR study suggested that the meta- and para-positions of the benzene ring of the phenylcarbamic amide moiety could be structurally modified by introducing halides or alkyl substituents.

Marine diterpenoid targets STING palmitoylation in mammalian cells.

Natural products are important sources of therapeutic agents and useful drug discovery tools. The fused macrocycles and multiple stereocenters of briarane-type diterpenoids pose a major challenge to total synthesis and efforts to characterize their biological activities. Harnessing a scalable source of excavatolide B (excB) from cultured soft coral Briareum stechei, we generated analogs by late-stage diversification and performed structure-activity analysis, which was critical for the development of functional excB probes. We further used these probes in a chemoproteomic strategy to identify Stimulator of Interferon Genes (STING) as a direct target of excB in mammalian cells. We showed that the epoxylactone warhead of excB is required to covalently engage STING at its membrane-proximal Cys91, inhibiting STING palmitoylation and signaling. This study reveals a possible mechanism-of-action of excB, and expands the repertoire of covalent STING inhibitors.

AKT phosphorylation as a predictive biomarker for PI3K/mTOR dual inhibition-induced proteolytic cleavage of mTOR companion proteins in small cell lung cancer.

BACKGROUND: Constitutive activation of PI3K signaling has been well recognized in a subset of small cell lung cancer (SCLC), the cancer type which has the most aggressive clinical course amongst pulmonary tumors. Whereas cancers that acquire a mutation/copy gain in PIK3CA or loss of PTEN have been implicated in enhanced sensitivity to inhibitors targeting the PI3K/AKT/mTOR pathway, the complexities of the pathway and corresponding feedback loops hamper clear predictions as to the response of tumors presenting these genomic features. METHODS: The correlation between the expression profile of proteins involved in the PI3K/AKT/mTOR signaling and cell viability in response to treatment with small molecule inhibitors targeting isoform-specific PI3Ks, AKT, and mTOR was assessed in 13 SCLC cancer cell lines. Athymic nude mice were used to determine the effect of PI3K/mTOR dual inhibition on the growth of xenograft SCLC tumors in vivo. The activation of caspase signaling and proteolytic cleavages of mTOR companion proteins were assessed using recombinant caspases assays and Western blot analyses. RESULTS: Our results indicate that the sensitivity of these SCLC cell lines to GSK2126458, a dual PI3K/mTOR inhibitor, is positively correlated with the expression levels of phosphorylated AKT (p-AKT) at Thr308 and Ser473. Inhibition of pan-class I PI3Ks or PI3K/mTOR dual inhibition was shown to induce proteolytic cleavage of RICTOR and RPTOR, which were respectively dependent on Caspase-6 and Caspase-3. A combination of a clinically approved PI3Kα-selective inhibitor and an mTORC1 inhibitor was shown to have synergistic effects in inducing the death of SCLC cells with high p-AKT. We observed no clear correlation between PTEN levels and the survival of SCLCs in response to PI3K/mTOR dual inhibition; however, PTEN depletion was shown to increase the susceptibility of low p-AKT SCLC cells to dual PI3K/mTOR inhibitor-induced cell death as well as the proteolytic cleavage of RICTOR. CONCLUSIONS: These results suggest the level of p-AKT can be a companion diagnostic biomarker for the treatment of SCLC involving the combinational use of clinically approved isoform-specific PI3K and mTOR inhibitors.

Deformation of the nucleus by TGFß1 via the remodeling of nuclear envelope and histone isoforms.

The cause of nuclear shape abnormalities which are often seen in pre-neoplastic and malignant tissues is not clear. In this study we report that deformation of the nucleus can be induced by TGFß1 stimulation in several cell lines including Huh7. In our results, the upregulated histone H3.3 expression downstream of SMAD signaling contributed to TGFß1-induced nuclear deformation, a process of which requires incorporation of the nuclear envelope (NE) proteins lamin B1 and SUN1. During this process, the NE constitutively ruptured and reformed. Contrast to lamin B1 which was relatively stationary around the nucleus, the upregulated lamin A was highly mobile, clustering at the nuclear periphery and reintegrating into the nucleoplasm. The chromatin regions that lost NE coverage formed a supra-nucleosomal structure characterized by elevated histone H3K27me3 and histone H1, the formation of which depended on the presence of lamin A. These results provide evidence that shape of the nucleus can be modulated through TGFß1-induced compositional changes in the chromatin and nuclear lamina.

Development of a respiratory syncytial virus vaccine using human hepatitis B core-based virus-like particles to induce mucosal immunity.

BACKGROUND: Respiratory syncytial virus (RSV) is an important viral pathogen responsible for severe infection of the lower respiratory tract in children under the age of 5 years. No vaccines against RSV are currently in clinical use. Vaccine-associated enhanced respiratory disease (ERD) caused by excess Th2 type responses was observed in a clinical trial of formalin-inactivated RSV (FI-RSV) in antigen-naïve infants. Thus, inducing a balanced immune response is a crucial issue in the development of an RSV vaccine. METHODS: In this study, we constructed, expressed, and purified a recombinant RSV vaccine candidate (i.e., HRØ24) containing the two heptad repeat regions and the antigenic sites Ø, II, and IV of the RSV F protein. The RSV vaccine candidate was intranasally administrated to BALB/c and C57BL/6 mice in combination with virus-like particles (VLPs) derived from the core protein of the hepatitis B virus (HBc). Mucosal immunity to HRØ24 was then assessed. RESULTS: Intranasal administration of HBc VLPs in combination with HRØ24 induced serum IgGs against HRØ24 as well as lung HRØ24-specific sIgAs in both C57BL/6 and BALB/c mouse models. The secretion of IFN-γ from splenocyte re-stimulation and an elevated ratio of serum IgG2a to IgG1 indicated that the immune response induced by the HBc VLPs/HRØ24 mixture was Th1-biased. Weight loss of <5% and no to low eosinophil infiltration was observed in histological analysis of the lung following a challenge with the RSV A2 strain. These results suggest that the HBc VLPs/HRØ24 combination conferred substantial partial protection against RSV-induced illness in mice. CONCLUSIONS: Long-term immunity to RSV-induced illness was achieved via intranasal vaccination using a mixture of HBc VLPs and HRØ24 in mouse models.

Discovery and Synthesis of a Pyrimidine-Based Aurora Kinase Inhibitor to Reduce Levels of MYC Oncoproteins.

The A-type Aurora kinase is upregulated in many human cancers, and it stabilizes MYC-family oncoproteins, which have long been considered an undruggable target. Here, we describe the design and synthesis of a series of pyrimidine-based derivatives able to inhibit Aurora A kinase activity and reduce levels of cMYC and MYCN. Through structure-based drug design of a small molecule that induces the DFG-out conformation of Aurora A kinase, lead compound 13 was identified, which potently (IC50 < 200 nM) inhibited the proliferation of high-MYC expressing small-cell lung cancer (SCLC) cell lines. Pharmacokinetic optimization of 13 by prodrug strategies resulted in orally bioavailable 25, which demonstrated an 8-fold higher oral AUC (F = 62.3%). Pharmacodynamic studies of 25 showed it to effectively reduce cMYC protein levels, leading to >80% tumor regression of NCI-H446 SCLC xenograft tumors in mice. These results support the potential of 25 for the treatment of MYC-amplified cancers including SCLC.

The SUN2-nesprin-2 LINC complex and KIF20A function in the Golgi dispersal.

The morphology of the Golgi complex is influenced by the cellular context, which strictly correlates with nuclear functions; however, the mechanism underlying this association remains elusive. The inner nuclear membrane SUN proteins, SUN1 and SUN2, have diverse functions together with the outer nuclear membrane nesprin proteins, which comprise the LINC complex. We found that depletion of SUN1 leads to Golgi complex dispersion with maintenance of ministacks and retained function for vesicle transport through the Golgi complex. In addition, SUN2 associates with microtubule plus-end-directed motor KIF20A, possibly via nesprin-2. KIF20A plays a role in the Golgi dispersion in conjunction with the SUN2-nesprin-2 LINC complex in SUN1-depleted cells, suggesting that SUN1 suppresses the function of the SUN2-nesprin-2 LINC complex under a steady-state condition. Further, SUN1-knockout mice, which show impaired cerebellar development and cerebellar ataxia, presented altered Golgi morphology in Purkinje cells. These findings revealed a regulation of the Golgi organization by the LINC complex.

Exercise and the Cisd2 Prolongevity Gene: Two Promising Strategies to Delay the Aging of Skeletal Muscle.

Aging is an evolutionally conserved process that limits life activity. Cellular aging is the result of accumulated genetic damage, epigenetic damage and molecular exhaustion, as well as altered inter-cellular communication; these lead to impaired organ function and increased vulnerability to death. Skeletal muscle constitutes ~40% of the human body's mass. In addition to maintaining skeletal structure and allowing locomotion, which enables essential daily activities to be completed, skeletal muscle also plays major roles in thermogenesis, metabolism and the functioning of the endocrine system. Unlike many other organs that have a defined size once adulthood is reached, skeletal muscle is able to alter its structural and functional properties in response to changes in environmental conditions. Muscle mass usually remains stable during early life; however, it begins to decline at a rate of ~1% year in men and ~0.5% in women after the age of 50 years. On the other hand, different exercise training regimens are able to restore muscle homeostasis at the molecular, cellular and organismal levels, thereby improving systemic health. Here we give an overview of the molecular factors that contribute to lifespan and healthspan, and discuss the effects of the longevity gene Cisd2 and middle-to-old age exercise on muscle metabolism and changes in the muscle transcriptome in mice during very old age.

Lamin A-mediated nuclear lamina integrity is required for proper ciliogenesis.

The primary cilium is a sensory organelle that receives specific signals from the extracellular environment important for vertebrate development and tissue homeostasis. Lamins, the major components of the nuclear lamina, are required to maintain the nuclear structure and are involved in most nuclear activities. In this study, we show that deficiency in lamin A/C causes defective ciliogenesis, accompanied by increased cytoplasmic accumulation of actin monomers and increased formation of actin filaments. Disruption of actin filaments by cytochalasin D rescues the defective ciliogenesis in lamin A/C-depleted cells. Moreover, lamin A/C-deficient cells display lower levels of nesprin 2 and defects in recruiting Arp2, myosin Va, and tau tubulin kinase 2 to the basal body during ciliogenesis. Collectively, our results uncover a functional link between nuclear lamina integrity and ciliogenesis and implicate the malfunction of primary cilia in the pathogenesis of laminopathy.

Progerin in muscle leads to thermogenic and metabolic defects via impaired calcium homeostasis.

Mutations in lamin A (LMNA) are responsible for a variety of human dystrophic and metabolic diseases. Here, we created a mouse model in which progerin, the lamin A mutant protein that causes Hutchinson-Gilford progeria syndrome (HGPS), can be inducibly overexpressed. Muscle-specific overexpression of progerin was sufficient to induce muscular dystrophy and alter whole-body energy expenditure, leading to premature death. Intriguingly, sarcolipin (Sln), an endoplasmic reticulum (ER)-associated protein involved in heat production, is upregulated in progerin-expressing and Lmna knockout (Lmna-/- ) skeletal muscle. The depletion of Sln accelerated the early death of Lmna-/- mice. An examination at the molecular level revealed that progerin recruits Sln and Calnexin to the nuclear periphery. Furthermore, progerin-expressing myoblasts presented enhanced store-operated Ca2+ entry, as well as increased co-localization of STIM1 and ORAI1. These findings suggest that progerin dysregulates calcium homeostasis through an interaction with a subset of ER-associated proteins, resulting in thermogenic and metabolic abnormalities.

Sun1 Mediates Interkinetic Nuclear Migration and Notch Signaling in the Neurogenesis of Zebrafish.

Interkinetic nuclear migration (INM) is a process by which nuclei oscillate between the basal and apical surfaces of epithelial cells in coordination with the cell cycle. The cytoskeletal machinery including microtubules and actin has been reported to drive apical INM; however, the role of nuclear proteins in this process has yet to be fully elucidated. Here, we investigated the function of a SUN-domain protein, Sun1, in zebrafish. We found that zebrafish sun1 is highly expressed in the ventricular zone of the brain. Knocking down sun1 with antisense morpholino oligonucleotides reduced the abundance of nestin- and gfap-expressing neural stem cells and progenitor cells. The live-cell imaging results showed that sun1 morphant cells migrated toward the basal side during the S phase but failed to migrate apically during the G2 phase. On the contrary, the passive stochastic movement during the G2 phase was unaffected. Furthermore, down regulation of sun1 was shown to reduce the expression of genes associated with the Notch pathway, whereas the expression of genes in the Wnt pathway was less perturbed. Findings from this research suggest that the Sun1-mediated nucleo-cytoskeletal interaction contributes to apical nuclear migration, and may thus affect exposure to Notch signal, thereby altering the composition of the progenitor pool in the embryonic neurogenesis of zebrafish.

Interference of DNAJB6/MRJ Isoform Switch by Morpholino Inhibits Replication of HIV-1 and RSV.

The molecular chaperon MRJ (DNAJB6) exhibits two splice isoforms that have different roles in human viral infection, but the regulatory mechanism of MRJ isoform expression is yet unclear. In this study, we show that reduction of the polyadenylation factor CstF64 was correlated with the increase of the MRJ large isoform (MRJ-L) in human macrophages and elucidate the mechanism underlying CstF64-modulated MRJ isoform expression. Moreover, we exploited an antisense strategy targeting MRJ-L for virus replication. A morpholino oligonucleotide complementary to the 5' splice site of MRJ intron 8 downregulated MRJ-L expression and suppressed the replication of not only HIV-1 but also respiratory syncytial virus (RSV). We demonstrated that downregulation of the MRJ-L level reduced HIV-1 replication as well as the subgenomic mRNA and viral production of RSV. The present findings that two human health-threatening viruses take advantage of MRJ-L for infection suggest MRJ-L as a potential target for broad-spectrum antiviral strategy.

Design and synthesis of BPR1K653 derivatives targeting the back pocket of Aurora kinases for selective isoform inhibition.

Twenty five novel chemical analogs of the previously reported Aurora kinase inhibitor BPR1K653 (1-(4-(2-((5-chloro-6-phenylfuro[2,3-d]pyrimidin-4-yl)amino)ethyl)phenyl)-3-(2-((dimethylamino)methyl)phenyl)urea) have been designed, synthesized, and evaluated by Aurora-A and Aurora-B enzymatic kinase activity assays. Similar to BPR1K653, analogs 3b-3h bear alkyl or tertiary amino group at the ortho position of the phenylurea, and showed equal or better inhibition activity for Aurora-B over Aurora-A. Conversely, preferential Aurora-A inhibition activity was observed when the same functional group was moved to the meta position of the phenylurea. Compounds 3m and 3n, both of which harbor a tertiary amino group at the meta position of the phenylurea, showed 10-16 fold inhibition selectivity for Aurora-A over Aurora-B. The in vitro kinase inhibition results were verified by Western blot analysis, and indicated that compounds 3m and 3n were more than 75-fold superior in inhibiting T-loop autophosphorylation of Aurora-A (Thr288), compared to Aurora-B (Thr232) in HCT116 colon carcinoma cells. The computational docking analysis suggested that the tertiary amine at the meta position of the phenylurea formed a more stable interaction with residues in the back pocket of Aurora-A than in Aurora-B, a possible explanation for the observed discrepancy in the selectivity. These results support an alternative small molecule design strategy targeting the back pocket of Aurora kinases for selective isoform inhibition.

Adaptation of influenza A (H7N9) virus in primary human airway epithelial cells.

Influenza A (H7N9) is an emerging zoonotic pathogen with pandemic potential. To understand its adaptation capability, we examined the genetic changes and cellular responses following serial infections of A (H7N9) in primary human airway epithelial cells (hAECs). After 35 serial passages, six amino acid mutations were found, i.e. HA (R54G, T160A, Q226L, H3 numbering), NA (K289R, or K292R for N2 numbering), NP (V363V/I) and PB2 (L/R332R). The mutations in HA enabled A(H7N9) virus to bind with higher affinity (from 39.2% to 53.4%) to sialic acid α2,6-galactose (SAα2,6-Gal) linked receptors. A greater production of proinflammatory cytokines in hAECs was elicited at later passages together with earlier peaking at 24 hours post infection of IL-6, MIP-1α, and MCP-1 levels. Viral replication capacity in hAECs maintained at similar levels throughout the 35 passages. In conclusion, during the serial infections of hAECs by influenza A(H7N9) virus, enhanced binding of virion to cell receptors with subsequent stronger innate cell response were noted, but no enhancement of viral replication could be observed. This indicates the existence of possible evolutional hurdle for influenza A(H7N9) virus to transmit efficiently from human to human.

Utrophin Compensates dystrophin Loss during Mouse Spermatogenesis.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder resulting from mutations in the dystrophin gene. The mdx/utrn -/- mouse, lacking in both dystrophin and its autosomal homologue utrophin, is commonly used to model the clinical symptoms of DMD. Interestingly, these mice are infertile but the mechanisms underlying this phenomenon remain unclear. Using dystrophin deficient mdx mouse and utrophin haplodeficient mdx/utrn +/- mouse models, we demonstrate the contribution of Dp427 (full-length dystrophin) and utrophin to testis and epididymis development, as well as spermatogenesis. We show that Dp427 deficiency disturbed the balance between proliferation and apoptosis of germ cells during spermatogenesis, which was further disrupted with utrophin haplodeficiency, deciphering a compensatory role of utrophin for dystrophin in the male reproductive system. In the spermatozoa, we have found a compensatory response of utrophin to dystrophin deficiency - namely the upregulation and relocation of utrophin to the flagellar midpiece. This study demonstrates the contribution of Dp427 and utrophin in male fertility, suggesting a potential pathology in DMD patients.

Outer nuclear membrane protein Kuduk modulates the LINC complex and nuclear envelope architecture.

Linker of nucleoskeleton and cytoskeleton (LINC) complexes spanning the nuclear envelope (NE) contribute to nucleocytoskeletal force transduction. A few NE proteins have been found to regulate the LINC complex. In this study, we identify one, Kuduk (Kud), which can reside at the outer nuclear membrane and is required for the development of Drosophila melanogaster ovarian follicles and NE morphology of myonuclei. Kud associates with LINC complex components in an evolutionarily conserved manner. Loss of Kud increases the level but impairs functioning of the LINC complex. Overexpression of Kud suppresses NE targeting of cytoskeleton-free LINC complexes. Thus, Kud acts as a quality control mechanism for LINC-mediated nucleocytoskeletal connections. Genetic data indicate that Kud also functions independently of the LINC complex. Overexpression of the human orthologue TMEM258 in Drosophila proved functional conservation. These findings expand our understanding of the regulation of LINC complexes and NE architecture.

In vivo inhibition of influenza A virus replication by RNA interference targeting the PB2 subunit via intratracheal delivery.

BACKGROUND: Influenza virus infection is a major threat to human health. Small interfering RNA (siRNA) is a promising approach for the prevention and treatment of viral infections. In this study, we constructed a series of DNA vector-based short hairpin RNAs (shRNAs) that target various genes of the influenza A virus using the polymerase III U6-RNA promoter to prevent influenza virus infection in vitro and in a mouse model. RESULTS: Three sets of DNA vector-based shRNA, two targeting genes encoding the polymerase acidic protein (PA) and one targeting polymerase basic protein 2 (PB2), efficiently inhibited the replication of influenza virus A/WSN/33(H1N1) in vitro. We also successfully prevented influenza virus A/WSN/33(H1N1) infection in a C57BL/6 mouse model by intratracheal delivery of anti-PB2 shRNA. CONCLUSIONS: Our findings suggest that the PB2-targeting shRNA plasmid showed potential for use as an RNAi-based therapeutic for influenza virus infection.

Neural stem cells promote nerve regeneration through IL12-induced Schwann cell differentiation.

Regeneration of injured peripheral nerves is a slow, complicated process that could be improved by implantation of neural stem cells (NSCs) or nerve conduit. Implantation of NSCs along with conduits promotes the regeneration of damaged nerve, likely because (i) conduit supports and guides axonal growth from one nerve stump to the other, while preventing fibrous tissue ingrowth and retaining neurotrophic factors; and (ii) implanted NSCs differentiate into Schwann cells and maintain a growth factor enriched microenvironment, which promotes nerve regeneration. In this study, we identified IL12p80 (homodimer of IL12p40) in the cell extracts of implanted nerve conduit combined with NSCs by using protein antibody array and Western blotting. Levels of IL12p80 in these conduits are 1.6-fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves motor recovery and increases the diameter up to 4.5-fold, at the medial site of the regenerated nerve. In vitro study further revealed that IL12p80 stimulates the Schwann cell differentiation of mouse NSCs through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that IL12p80 can trigger Schwann cell differentiation of mouse NSCs through Stat3 phosphorylation and enhance the functional recovery and the diameter of regenerated nerves in a mouse sciatic nerve injury model.