Human Homologue Research Articles

Dual Acting Immuno-Antibiotics: Computational Investigation on Antibacterial Efficacy of Immune Boosters Against Isoprenoid H Enzyme.

Drug-resistant infections have become a serious threat to human health in the past two decades. Global Antimicrobial Surveillance (GLASS) in January 2018 reported widespread antibiotic resistance among 1.5 million people infected with bacteria across 22 countries. According to prominent economist Jim O'Neil, antimicrobial resistance is estimated to kill ∼10 million people affected by microorganisms each year by 2050. Even though multiple therapeutics are now available to treat the infections, more and more bacterial strains have acquired resistance to these treatments through various techniques. Moreover, the decrease in the pipeline of antibacterial medicines under clinical development has become a significant problem. In this scenario, the development of novel antibiotics that act on untapped pathways is necessary to combat the bacterial infections. Isoprenoid H (IspH) synthetase has become an attractive antibacterial target as there is no human homologue. IspH is an enzyme involved in methyl-d-erythritol phosphate (MEP) pathway of isoprenoid synthesis and is conserved in gram-negative bacteria, mycobacteria, and apicomplexans. Since, IspH is a novel therapeutic target, explorations are only just beginning, and despite the progress made in this area, no single IspH inhibitor is available in the market for therapeutic use. In this article, we have repurposed 35 immune boosters against IspH enzyme using methods such as extra-precision docking and Molecular Mechanics Generalized Born Surface Area (MMGBSA). Among them, 4'-fluorouridine was found to be active because of its glide score and significant binding affinity with IspH enzyme. Furthermore, this study requires more in vitro, in vivo, and molecular dynamics studies to support our findings.

Structural dynamics and in silico design of pyrazolopyran-based inhibitors against Plasmodium serine hydroxymethyltransferases

The clinical efficacy of antimalarial drugs has been reduced due to resistance spreading over many parts of the world. Target-based approaches on attractive drug targets, such as Plasmodium serine hydroxymethyltransferases (SHMTs) exhibiting distinct structure and function as well as kinetic mechanisms from the human enzyme homologue, are highly useful methods to be used for bypassing the present resistance in the field. Herein, 500-ns molecular dynamics (MD) simulations were carried out to investigate the mode of action of pyrazolopyran(+)-85 and pyrazolopyran(+)-86 with the most attractive inhibition efficiency in Plasmodium falciparum and P. vivax SHMTs (in the Schiff base form of PLP-L-serine (PLS) bound enzyme). The binding free energy results indicated the binding affinity of pyrazolopyran(+)-86 to Plasmodium SHMTs that is more favorable than pyrazolopyran(+)-85 by ∼ 2 kcal⋅mol−1, supported by the stronger ligand–protein hydrogen bonding and the lower solvent accessibility within the enzyme active site. According to the per-residue decomposition free energy analysis, residues L124, G128, H129, L130, K139, N356, and T357 are essential for inhibitors binding. By the rational structure-based drug design, the isopropyl moiety on the pyrazolopyran core should be changed to the negatively charged group (e.g., carboxylate group) for interacting with the positively charged residue R371. Alternatively, the phenolic compounds could be substituted with a phenyl or piperidine ring to promote hydrogen bond formation with the surrounding residues. Therefore, our findings presented here provide insights into the mode of inhibition of pyrazolopyran-based inhibitors and rational ideas for designing novel antimalarial drugs targeting Plasmodium SHMTs.

Identification of healthspan-promoting genes in Caenorhabditis elegans based on a human GWAS study

To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn’s disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly.

G1-Cyclin2 (Cln2) promotes chromosome hypercondensation in eco1/ctf7 rad61 null cells during hyperthermic stress in Saccharomyces cerevisiae.

Eco1/Ctf7 is a highly conserved acetyltransferase that activates cohesin complexes and is critical for sister chromatid cohesion, chromosome condensation, DNA damage repair, nucleolar integrity, and gene transcription. Mutations in the human homolog of ECO1 (ESCO2/EFO2), or in genes that encode cohesin subunits, result in severe developmental abnormalities and intellectual disabilities referred to as Roberts syndrome and Cornelia de Lange syndrome, respectively. In yeast, deletion of ECO1 results in cell inviability. Codeletion of RAD61 (WAPL in humans), however, produces viable yeast cells. These eco1 rad61 double mutants, however, exhibit a severe temperature-sensitive growth defect, suggesting that Eco1 or cohesins respond to hyperthermic stress through a mechanism that occurs independent of Rad61. Here, we report that deletion of the G1 cyclin CLN2 rescues the temperature-sensitive lethality otherwise exhibited by eco1 rad61 mutant cells, such that the triple mutant cells exhibit robust growth over a broad range of temperatures. While Cln1, Cln2, and Cln3 are functionally redundant G1 cyclins, neither CLN1 nor CLN3 deletions rescue the temperature-sensitive growth defects otherwise exhibited by eco1 rad61 double mutants. We further provide evidence that CLN2 deletion rescues hyperthermic growth defects independent of START and impacts the state of chromosome condensation. These findings reveal novel roles for Cln2 that are unique among the G1 cyclin family and appear critical for cohesin regulation during hyperthermic stress.

Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.

DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.

Experiment verification test of the Artemis I ‘Deep Space Radiation Genomics’ experiment

When the Artemis missions launch, NASA's Orion spacecraft (and crew as of the Artemis II mission) will be exposed to the deep space radiation environment beyond the protection of Earth's magnetosphere. Hence, it is essential to characterize the effects of space radiation, microgravity, and the combination thereof on cells and organisms, i.e., to quantify any correlations between the deep space radiation environment, genetic variation, and induced genetic changes in cells. To address this, the Artemis I mission will include the Peristaltic Laboratory for Automated Science with Multigenerations (PLASM) hardware containing the Deep Space Radiation Genomics (DSRG) experiment. The scientific aims of DSRG are (i) to identify the metabolic and genomic pathways in yeast affected by microgravity, space radiation, and their combination, and (ii) to differentiate between gravity and radiation exposure on single-gene deletion/overexpressing strains' ability to thrive in the spaceflight environment. Yeast is used as a model system because 70% of its essential genes have a human homolog, and over half of these homologs can functionally replace their human counterpart. As part of the experiment preparation towards spaceflight, an Experiment Verification Test (EVT) was performed at the Kennedy Space Center to verify that the experiment design, hardware, and approach to automated operations will enable achieving the scientific aims. For the EVT, fluidic systems were assembled, sterilized, loaded, and acceptance-tested, and subsequently integrated with the engineering parts to produce a flight-like PLASM unit. Each fluidic system consisted of (i) a Media Bag, (ii) four Culture Bags loaded with Saccharomyces cerevisiae (two with deletion series and the remaining two with overexpression series), and (iii) tubing and check valves. The EVT PLASM unit was put under a temperature profile replicating the anticipated different phases of flight, including handover to launch, spaceflight, and splashdown to handover back to the science team, for a 58-day period. At EVT completion, the rate of activation, cellular growth, RNA integrity, and sample contamination were interrogated. All of the experiment's success criteria were satisfied, encouraging our efforts to perform this investigation on Artemis I. This manuscript thus describes the process of spaceflight experiment design maturation with a focus on the EVT, its results, DSRG's preparation for its planned launch on Artemis I in 2022, and how the PLASM hardware can enable other scientific goals on future Artemis missions and/or the Lunar Orbital Platform – Gateway.

Abstract 799: HLTF and SHPRH in mismatch repair and cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer deaths, with an increasing rate of CRC diagnosis in younger individuals. MMR is the DNA repair mechanism that repairs DNA mispairs and small insertions or deletions remaining after replication. MMR is also required for apoptosis after certain types of exogenous DNA damage that result in damage-associated mispairs. MMR defects are the underlying cause of Lynch syndrome, a familial cancer predisposition syndrome that increases susceptibility to multiple cancers, specifically colorectal cancer. MMR defects are also commonly found in sporadic colorectal cancers. Model systems such as Saccharomyces cerevisiae, Escherichia coli, and human cell lines have been used to study the MMR proteins and pathways. The basics of the MMR mechanism are fairly well understood; however, proteins associated with MMR are still being identified, and the roles of these proteins are largely unknown. We have identified the yeast protein Rad5 as an interactor with the yeast MMR proteins Msh2 and Mlh1. Rad5 is a helicase and an E3 ubiquitin ligase which is involved in post-replicative repair and damage tolerance. However, to date, Rad5 has no known role in MMR. We have determined that these interactions are conserved through evolution to human Rad5 homologs, HLTF and SHPRH. The Rad5 interactions with Mlh1 and Msh2 appear to be split between the human homologs with human Msh2 interacting with HLTF and human Mlh1 interacting with SHPRH. We have found that loss of SHPRH induces resistance to MMR-mediated apoptosis. Current experiments are in progress to determine the binding sites between these proteins. We are also investigating what functional impact the Rad5 homologs have on mutation rate and MMR-induced apoptosis and how the interactions affect the roles of MMR. Together this will allow for a deeper understanding of how accessory proteins may influence canonical and non-canonical MMR. Since MMR status is currently used to determine patient treatment, understanding how commonly mutated accessory factors interact is critical. Citation Format: Anna Kristin Miller, Guogen Mao, Christine Rahal, Christopher Putnam, Eva Goellner. HLTF and SHPRH in mismatch repair and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 799.

Abstract SY39-01: The DNA damageome and cancer evolution

Abstract DNA damage provokes mutations, genome instability, cancer and biological aging and results from external agents or endogenous cellular processes. We identified networks of unrelated proteins of diverse functions that promote DNA damage when overproduced: the DNA “damageome” proteins (DDPs). We found a large overproduction DDP network in Escherichia coli and find that the proteins promote mutagenesis and genome instability by various mechanisms—three of which we identify. Their 284 human homologs are over-represented among known cancer drivers, and their expression in tumors predicts heavy mutagenesis and poor prognosis. When overproduced in human cells, many of the human homologs promote DNA damage and mutation, with DNA-damaging mechanisms like those in E. coli. As the earliest upstream provocateurs of cancer evolution, the proteins of DNA damageome networks may indicate that there are evolution-accelerating functions of many human known and newly implicated cancer-promoting proteins. Citation Format: Susan Rosenberg. The DNA damageome and cancer evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY39-01.

Abstract 2421: Elucidation of CSF1 autocrine signaling mediated by mutant p53 in metastatic esophageal carcinoma

Abstract Introduction: Esophageal cancer is a highly aggressive cancer and is the 6th leading cause of cancer mortality worldwide, with esophageal squamous cell carcinoma (ESCC) being the most prevalent subtype. Mutations in the tumor suppressor TP53 (human homolog of mouse Trp53), which encodes p53, are detected frequently in ESCC that in turn correlate with poor survival and high metastatic rates. We have conducted RNA-Seq and cytokine array on primary and metastatic tumor cells harvested from our mouse models with Trp53R172H mutation. We have identified colony stimulating factor 1 (CSF1) to be upregulated. Our data suggest the existence of CSF1-CSF1R autocrine signaling, and its functional relationship to the promotion of metastasis in ESCC, recognizing that CSF1 is known to be critical in the polarization of macrophages to M2-like tumor associated macrophages. Our goal is to investigate the role and mediators of CSF1/CSF1R signaling by which missense TP53 mutations can promote invasion and lung metastasis in ESCC. Methods: We used L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and 2D/3D cell culture systems to model ESCC. To assess the functional role of CSF1, we established a CRISPR approach to knockout Csf1in our cells introducing ribonucleoprotein (RNP) Cas9/gRNA complex via nucleofection and overexpressed Csf1. We also utilized a small molecule inhibitor of CSF1R, GW2580. We conducted TCGA analysis on the association of distinct TP53 mutations with ESCC prognosis and Csf1 expression, as well as Csf1 expression in metastatic tumors. Based on this, we modeled ESCC by introducing various DNA contact and conformational p53 mutations into esophageal cells with wildtype p53 via base editing. Results: TCGA data and our models demonstrate that metastatic ESCCs have increased Csf1 expression compared to primary tumors and it is dependent upon p53 mutation status. In addition, analysis of ESCC patients’ datasets reveal that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. We show that Csf1 function is related to tumor cell invasion, primary tumor growth and metastatic tumor burden in a mutant p53 background. We have identified both bromodomain and extra-terminal motif (BET) protein BRD4 and transcription factor nuclear factor kappa B (NF-κB) as proteins that interact with Trp53R172H at higher levels compared to wildtype p53 to induce Csf1 expression. In addition, inhibition of CSF1R with small molecule GW2580 downregulated JAK-STAT3 pathway mediating epithelial-mesenchymal transition (EMT) mechanisms that would contribute to increased invasiveness and metastasis. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R autocrine signaling and its mediators in promoting lung metastasis from ESCC that may be applicable to other squamous cell cancers. We believe this mechanism can be targeted therapeutically. Citation Format: Gizem Efe, Qiaosi Tang, Ricardo Cruz-Acuna, Kensuke Suzuki, Kensuke Sugiura, Joel Gabre, Uma Sachdeva, Wei Gu, Carol L. Prives, Anil K. Rustgi. Elucidation of CSF1 autocrine signaling mediated by mutant p53 in metastatic esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2421.

An In Vivo Model of Echovirus-Induced Meningitis Defines the Differential Roles of Type I and Type III Interferon Signaling in Central Nervous System Infection.

Echoviruses are among the most common worldwide causes of aseptic meningitis, which can cause long-term sequelae and death, particularly in neonates. However, the mechanisms by which these viruses induce meningeal inflammation are poorly understood, owing at least in part to the lack of in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we developed an in vivo neonatal mouse model that recapitulates key aspects of echovirus-induced meningitis. We show that expression of the human homologue of the primary echovirus receptor, the neonatal Fc receptor (FcRn), is not sufficient for infection of the brains of neonatal mice. However, ablation of type I, but not III, interferon (IFN) signaling in mice expressing human FcRn permitted high levels of echovirus replication in the brain, with corresponding clinical symptoms, including delayed motor skills and hind-limb weakness. Using this model, we defined the immunological response of the brain to echovirus infection and identified key cytokines, such as granulocyte colony-stimulating factor (G-CSF) and interleukin 6 (IL-6), that were induced by this infection. Lastly, we showed that echoviruses specifically replicate in the leptomeninges, where they induce profound inflammation and cell death. Together, this work establishes an in vivo model of aseptic meningitis associated with echovirus infections that delineates the differential roles of type I and type III IFNs in echovirus-associated neuronal disease and defines the specificity of echoviral infections within the meninges. IMPORTANCE Echoviruses are among the most common worldwide causes of aseptic meningitis, which can cause long-term sequelae or even death. The mechanisms by which echoviruses infect the brain are poorly understood, largely owing to the lack of robust in vivo models that recapitulate this aspect of echovirus pathogenesis. Here, we establish a neonatal mouse model of echovirus-induced aseptic meningitis and show that expression of the human homologue of the FcRn, the primary receptor for echoviruses, and ablation of type I IFN signaling are required to recapitulate echovirus-induced meningitis and clinical disease. These findings provide key insights into the host factors that control echovirus-induced meningitis and a model that could be used to test anti-echovirus therapeutics.

1H, 13C, and 15N resonance assignments and solution structures of the KH domain of human ribosome binding factor A, mtRbfA, involved in mitochondrial ribosome biogenesis.

Ribosome biogenesis is a complicated, multistage process coordinated by ribosome assembly factors. Ribosome binding factor A (RbfA) is a bacterial one, which possesses a single structural type-II KH domain. By this domain, RbfA binds to a 16S rRNA precursor in small ribosomal subunits to promote its 5'-end processing. The human RbfA homolog, mtRbfA, binds to 12S rRNAs in the mitoribosomal small subunits and promotes its critical maturation process, the dimethylation of two highly conserved consecutive adenines, which differs from that of RbfA. However, the structural basis of the mtRbfA-mediated maturation process is poorly understood. Herein, we report the 1H, 15N, and 13C resonance assignments of the KH domain of mtRbfA and its solution structure. The mtRbfA domain adopts essentially the same α1-β1-β2-α2(kinked)-β3 topology as the type-II KH domain. Comparison with the RbfA counterpart showed structural differences in specific regions that function as a putative RNA-binding site. Particularly, the α2 helix of mtRbfA forms a single helix with a moderate kink at the Ser-Ala-Ala sequence, whereas the corresponding α2 helix of RbfA is interrupted by a distinct kink at the Ala-x-Gly sequence, characteristic of bacterial RbfA proteins, to adopt an α2-kink-α3 conformation. Additionally, the region linking α1 and β1 differs considerably in the sequence and structure between RbfA and mtRbfA. These findings suggest some variations of the RNA-binding mode between them and provide a structural basis for mtRbfA function in mitoribosome biogenesis.

Conformational dynamics in the disordered region of human CPEB3 linked to memory consolidation

BackgroundCurrent understanding of the molecular basis of memory consolidation points to an important function of amyloid formation by neuronal-specific isoforms of the cytoplasmic polyadenylation element binding (CPEB) protein family. In particular, CPEB is thought to promote memory persistence through formation of self-sustaining prion-like amyloid assemblies at synapses, mediated by its intrinsically disordered region (IDR) and leading to permanent physical alterations at the basis of memory persistence. Although the molecular mechanisms by which amyloid formation takes place in CPEB have been described in invertebrates, the way amyloid formation occurs in the human homolog CPEB3 (hCPEB3) remains unclear. Here, we characterize by NMR spectroscopy the atomic level conformation and ps-ms dynamics of the 426-residue IDR of hCPEB3, which has been associated with episodic memory in humans.ResultsWe show that the 426-residue N-terminal region of hCPEB3 is a dynamic, intrinsically disordered region (IDR) which lacks stable folded structures. The first 29 residues, M1QDDLLMDKSKTQPQPQQQQRQQQQPQP29, adopt a helical + disordered motif, and residues 86–93: P83QQPPPP93, and 166–175: P166PPPAPAPQP175 form polyproline II (PPII) helices. The (VG)5 repeat motif is completely disordered, and residues 200–250 adopt three partially populated α-helices. Residues 345–355, which comprise the nuclear localization signal (NLS), form a modestly populated α-helix which may mediate STAT5B binding. These findings allow us to suggest a model for nascent hCPEB3 structural transitions at single residue resolution, advancing that amyloid breaker residues, like proline, are a key difference between functional versus pathological amyloids.ConclusionOur NMR spectroscopic analysis of hCPEB3 provides insights into the first structural transitions involved in protein–protein and protein-mRNA interactions. The atomic level understanding of these structural transitions involved in hCPEB3 aggregation is a key first step toward understanding memory persistence in humans, as well as sequence features that differentiate beneficial amyloids from pathological ones.AreasBiophysics, Structural Biology, Biochemistry & Neurosciences.

Zebrafish Klf11b is Required to Maintain Cell Viability by Inhibiting p53-Mediated Apoptosis.

Krüppel-like factor 10 (KLF10) regulates various cellular functions, such as proliferation, differentiation and apoptosis, as well as the homeostasis of several types of tissue. In the present study, we attempted a loss-of-function analysis of zebrafish Klf11a and Klf11b, which constitute human KLF10 homologs. Embryos injected with klf11b-morpholino (MO) showed developmental retardation and cell death, whereas klf11a-MO-injected embryos showed normal development. In klf11b-MO-injected embryos, a dramatic increase in the amount of zebrafish p53 mRNA might be the cause of the increase in that of bax. The degree of apoptosis decreased in the klf11b-MO and p53-MO co-injected embryos. These findings imply that KLF10 is a negative regulator of p53-dependent transcription, suggesting that the KLF10/p53 complex may play an important role in apoptosis for maintenance of tissue homeostasis during embryonic development.

A phase II study, with a safety lead-in, to evaluate ATX-101, a peptide drug targeting PCNA, in advanced dedifferentiated liposarcoma and leiomyosarcoma.

TPS11587 Background: Soft tissue sarcoma (STS) is a heterogeneous malignancy of mesenchymal origin; leiomyosarcoma (LMS) and liposarcoma (LPS), two of the most common adult STS, are treated with first-line chemotherapy with objective response rates (ORRs) of 15-20%. There is an urgent need for novel therapeutics. ATX-101 is a small peptide comprised of a novel human AlkB homolog 2 proliferating cell nuclear antigen (PCNA) interacting motif termed APIM coupled to cellular and nuclear delivery domains. Via APIM, PCNA interacts with many cellular proteins important in the cellular stress and DNA damage responses, as well as intracellular signaling, apoptosis, metabolism, and anti-tumor immunity. In preclinical studies, ATX-101 demonstrated single-agent activity and potentiated other cytotoxic and targeted agents across multiple cancer models in vitro and in vivo, including LMS and LPS. In a phase I safety and pharmacokinetic study in solid tumors, ATX-101 was well tolerated and demonstrated prolonged disease stabilization in patients (pts) with heavily pretreated malignancies. This study will evaluate preliminary efficacy and further establish the safety profile of ATX-101 in advanced LMS and LPS. Methods: This is a single-arm, open-label, Simon 2-stage, phase II clinical trial of ATX-101 in pts with advanced LMS and LPS. Eligible pts have ECOG PS ≤2, progression on ≥1 prior line of therapy and disease measurable by RECIST v1.1 and amenable to image-guided biopsy. Pts receive ATX-101 60 mg/m2 IV weekly in continuous 21-day cycles. The 1° endpoint is progression free rate at 12 weeks (PFR12). A Simon 2-stage design is used to evaluate for improvement in PFR12 of ≤ 30% (null hypothesis) versus ≥ 55% (alternative hypothesis). The design calls for 34 pts with a safety lead-in among the first 10 pts enrolled. If 15/34 meet the PFR12 endpoint, the treatment is promising. This design yields 85% power and 1-sided type I error of 5%. 2° endpoints include progression free survival, ORR, and safety. 10 pts undergo tumor biopsies pre-treatment and during cycle 2. Tissue is used for correlative analysis interrogating ATX-101’s effects on the immune microenvironment through multiplex immunohistochemistry, DNA damage response through whole exome sequencing/RNAseq to evaluate for alterations in HR pathway component genes, and intracellular signaling pathways by Western blot for AKT/mTOR components. The study opened to accrual 12/2021. Clinical trial information: NCT05116683.

Mitochondrial phosphatidylethanolamine synthesis affects mitochondrial energy metabolism and quiescence entry through attenuation of Snf1/AMPK signaling in yeast.

The Ups2-Mdm35 complex mediates intramitochondrial phosphatidylserine (PS) transport to facilitate mitochondrial phosphatidylethanolamine (PE) synthesis. In the present study, we found that ups2∆ yeast showed increased mitochondrial ATP production and enhanced quiescence (G0) entry in the post-diauxic shift phase. Transcriptomic and biochemical analyses revealed that the depletion of Ups2 leads to overactivation of the yeast AMPK homolog Snf1. Inactivation of Snf1 by depletion of an Snf1-activating kinase, Sak1 canceled the changes in mitochondrial ATP production and quiescence entry observed in ups2∆ cells. Furthermore, among the factors regulated by Snf1, upregulation of pyruvate carboxylase, Pyc1 and downregulation of acetyl-CoA carboxylase, Acc1, respectively, were sufficient to increase mitochondrial ATP production and quiescence entry. These results suggested that a normal PE synthesis mediated by Ups2-Mdm35 complex attenuates Snf1/AMPK activity, and that Snf1-mediated regulation of carbon metabolisms has great impacts on mitochondrial energy metabolism and quiescence entry. We also found that depletion of Ups2 together with the cell-cycle regulators Whi5 and Whi7, functional orthologs of the Rb1 tumor suppressor, caused a synthetic growth defect in yeast. Similarly, knockdown of PRELID3b, the human homolog of Ups2, decreased the viability of Rb1-deficient breast cancer cells, suggesting that PRELID3b is a potential target for cancer therapy.

Therapeutical Significance of Serpina3n Subsequent Cerebral Ischemia via Cytotoxic Granzyme B Inactivation.

Ischemic stroke is a devastating CNS insult with few clinical cures. Poor understanding of underlying mechanistic network is the primary limitation to develop novel curative therapies. Extracellular accumulation of granzyme B subsequent ischemia promotes neurodegeneration. Inhibition of granzyme B can be one of the potent strategies to mitigate neuronal damage. In present study, we investigated the effect of murine Serpina3n and human (homolog) SERPINA3 against cerebral ischemia through granzyme B inactivation. Recombinant Serpina3n/SERPINA3 were expressed by transfected 293 T cells, and eluted proteins were examined for postischemic influence both in vitro and in vivo. During in vitro test, Serpina3n was found effective enough to inhibit granzyme B, while SERPINA3 was ineffectual to counter cytotoxic protease. Treatment of hypoxic culture with recombinant Serpina3n/SERPINA3 significantly increased cell viability in dosage-dependent manner, recorded maximum at the highest concentration (4 mM). Infarct volume analysis confirmed that 50 mg/kg dosage of exogenous Serpina3n was adequate to reduce disease severity, while SERPINA3 lacked behind in analeptic effect. Immunohistochemical test, western blot analysis, and protease activity assay's results illustrated successful diffusion of applied protein to the ischemic lesion and reactivity with the target protease. Taken together, our findings demonstrate therapeutic potential of Serpina3n by interfering granzyme B-mediated neuronal death subsequent cerebral ischemia.

Molecular Modelling and Virtual Screening to Identify New Piperazine Derivatives as Potent Human 5-HT1A Antagonists and Reuptake Inhibitors.

Serotonin/5-HT antagonist and reuptake inhibitors (SARIs) ameliorate depression by increasing the terminal 5-HT through the activation of somatodendritic 5-HT1A autoreceptors. In addition to their therapeutic application as standalone antidepressants, they are co-administered with selective serotonin reuptake inhibitors (SSRI) to improve unpleasant side effects associated with SSRI-treated depression. However, only a few of the atypical antidepressants are available and not without some serious aftereffects. This study aims at the identification of novel promising SARIs using computational chemistry and high throughput screening. Pharmacophore features were modelled using LigandScout 4.3 and validated through the area under curve (AUC), enrichment factor (EF) and Guner-Henry (GH) scores. Molecular docking was employed for virtual screening against modelled human 5HT1A homology receptor, molecular dynamics simulations and ADMET predictions. The adopted pharmacophore possesses AUC, EF and GH scores of 0.7, 30.9 and 0.6 respectively, thus validated and used for molecular database screening. The modelled 5-HT1A homology receptor, validated using RCSB structure validation protocols, was employed for molecular docking and dynamics simulations. From the IBScreen database, the ligands, STOCK6S-36853, STOCK7S-36094, STOCK3S-94557, STOCK7S-28769 and STOCK5S-36248 interacted more strongly against the 5-HT1A receptor with docking scores of -8.735, -8.677, -8.140, -7.911 and -7.710 kcal/mol, and binding free energy of -29.72, -38.87, -29.85, -7.65 and -34.71 kcal/mol respectively, compared to fluoxetine and trazodone (positive controls) while albendazole and metformin (negative controls) scored least. They demonstrated good stability, satisfy the BDDCS RO5 and thus, are identified as potent SARIs. The study represents a cost-effective, faster and environmentally friendly approach to the discovery of promising SARI antidepressants for further translational study.

Conservative analysis of Synaptopodin-2 intron sense-overlapping lncRNA reveals its novel function in promoting muscle atrophy.

BackgroundDissection of the regulatory pathways that control skeletal muscle development and atrophy is important for the treatment of muscle wasting. Long noncoding RNA (lncRNA) play important roles in various stages of muscle development. We previously reported that Synaptopodin‐2 (SYNPO2) intron sense‐overlapping lncRNA (SYISL) regulates myogenesis through an interaction with enhancer of zeste homologue 2 (EZH2). However, it remains unclear whether SYISL homologues exist in humans and pigs, and whether the functions and mechanisms of these homologues are conserved among species.MethodsBioinformatics, cell fractionation, and quantitative real‐time polymerase chain reaction (qRT‐PCR) analyses were used for the identification and molecular characterization of SYISL homologues in humans and pigs. Effects on myogenesis and muscle atrophy were determined via loss‐of‐function or gain‐of‐function experiments using C2C12 myoblasts, myogenic progenitor cells, dexamethasone (DEX), and aging‐induced muscle atrophy models. RNA pulldown, RNA immunoprecipitation, dual luciferase reporting, and co‐transfection experiments were used to explore the mechanisms of SYISL interactions with proteins and miRNAs.ResultsWe identified SYISL homologues in humans (designated hSYISL) and pigs (designated pSYISL). Functional experiments demonstrated that hSYISL and pSYISL regulate myogenesis through interactions with EZH2. Interestingly, we showed that SYISL functions to regulate muscle atrophy and sarcopenia through comparative analysis. SYISL is significantly up‐regulated after muscle atrophy (P < 0.01); it significantly promotes muscle atrophy in DEX‐induced muscle atrophy models (P < 0.01). SYISL knockdown or knockout alleviates muscle atrophy and sarcopenia in DEX‐induced and aged mice. The tibialis anterior (TA) muscle weight of 3‐month‐old wild‐type (WT) mice decreased by 33.24% after DEX treatment (P < 0.001), while the muscle weight loss of 3‐month‐old SYISL knockout mice was only 18.20% after DEX treatment (P < 0.001). SYISL knockout in 18‐month‐old WT mice significantly increased the weights of quadriceps (Qu), gastrocnemius (Gas), and TA muscles by 10.45% (P < 0.05), 13.95% (P < 0.01), and 24.82% (P < 0.05), respectively. Mechanistically, SYISL increases the expression levels of the muscle atrophy genes forkhead box protein O3a (FoxO3a), muscle ring finger 1 (MuRF1), and muscle atrophy‐related F‐box (Atrogin‐1) via sponging of miR‐23a‐3p/miR‐103‐3p/miR‐205‐5p and thus promotes muscle atrophy. Additionally, we verified that human SYISL overexpression in muscles of 18‐month‐old WT mice significantly decreased the weights of Gas, Qu, and TA muscles by 7.76% (P < 0.01), 12.26% (P < 0.05), and 13.44% (P < 0.01), respectively, and accelerates muscle atrophy through conserved mechanisms.ConclusionsOur results identify SYISL as a conserved lncRNA that modulates myogenesis in mice, pigs, and humans. We also demonstrated its previously unknown ability to promote muscle atrophy.

CDK4/6 inhibitors sensitize gammaherpesvirus-infected tumor cells to T-cell killing by enhancing expression of immune surface molecules

BackgroundThe two oncogenic human gammaherpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein–Barr virus (EBV), both downregulate immune surface molecules, such as MHC-I, ICAM-1, and B7-2, enabling them to evade T-cell and natural killer cell immunity. Both also either encode for human cyclin homologues or promote cellular cyclin activity, and this has been shown to be important for proliferation and survival of gammaherpesvirus-induced tumors. CDK4/6 inhibitors, which are approved for certain breast cancers, have been shown to enhance expression of MHC-I in cell lines and murine models of breast cancer, and this was attributed to activation of interferons by endogenous retrovirus elements. However, it was not known if this would occur in gammaherpesvirus-induced tumors in which interferons are already activated.MethodsMultiple KSHV/EBV-infected cell lines were treated with CDK4/6 inhibitors. The growth of viable cells and expression of surface markers was assessed. T cell activation stimulated by the treated cells was assayed by a T-cell activation bioassay. Both viral and host gene expression was surveyed using RT-qPCR.ResultsThree CDK4/6 inhibitors, abemaciclib, palbociclib, and ribociclib, inhibited cell growth in KSHV-induced primary effusion lymphoma (PEL) and EBV positive Burkitt’s lymphoma (BL) cell lines, and KSHV-infected human umbilical vein endothelial cells (HUVECs). Moreover, CDK4/6 inhibitors increased mRNA and surface expression of MHC-I in all three and prevented downregulation of MHC-I surface expression during lytic replication in KSHV-infected cells. CDK4/6 inhibitors also variably increased mRNA and surface expression of ICAM-1 and B7-2 in the tested lines. Abemaciclib also significantly enhanced T-cell activation induced by treated PEL and BL cells. Certain gammaherpesvirus genes as well as endogenous retrovirus (ERV) 3–1 genes were enhanced by CDK4/6 inhibitors in most PEL and BL lines and this enhancement was associated with expression of gamma interferon-induced genes including MHC-I.ConclusionsThese observations provide evidence that CDK4/6 inhibitors can induce expression of surface immune markers MHC-I, B7-2, and ICAM-1 in gammaherpesvirus-infected cell lines and induce virus-specific immunity. They can thus thwart virus-induced immune evasion. These effects, along with their direct effects on KSHV- or EBV-induced tumors, provide a rational for the clinical testing of these drugs in these tumors.

Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport

The molecular mechanisms underlying the anterograde surface transport of G protein–coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α2A-adrenergic receptor (α2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER–Golgi-surface transport of α2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β2-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.