Dibenzofuran Derivatives Inspired from Cercosporamide as Dual Inhibitors of Pim and CLK1 Kinases

Pim kinases (proviral integration site for Moloney murine leukemia virus kinases) are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Thus, Pim kinases are validated as targets for antitumor therapy. In this context, our combined efforts in natural product-inspired library generation and screening furnished very promising dibenzo[b,d]furan derivatives derived from cercosporamide. Among them, lead compound 44 was highlighted as a potent Pim-1/2 kinases inhibitor with an additional nanomolar IC50 value against CLK1 (cdc2-like kinases 1) and displayed a low micromolar anticancer potency towards the MV4-11 (AML) cell line, expressing high endogenous levels of Pim-1/2 kinases. The design, synthesis, structure–activity relationship, and docking studies are reported herein and supported by enzyme, cellular assays, and Galleria mellonella larvae testing for acute toxicity.

Comparative evaluation of BMI-1 proto-oncogene expression in normal tissue, adenoma and papillary carcinoma of human thyroid in pathology samples

Objective Papillary Thyroid carcinoma accounts for more than 60% of adult thyroid carcinomas. Finding a helpful marker is vital to determine the correct treatment approach. The present study was aimed to evaluate the expression of the B cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) gene in papillary carcinoma, adenoma, and adjacent healthy thyroid tissues. Pathology blocks of thyroid tissues at the pathology department of patients who have undergone thyroid surgery between 2015 and 2019 were examined; papillary carcinoma, adenoma, and healthy tissues were selected and sectioned. Total RNA was extracted, and the relative expression level of the BMI-1 gene was examined using the Real-Time qPCR method. Results In the papillary and adenoma tissues, BMI-1 was overexpressed (1.047-fold and 1.042-fold) in comparison to healthy tissues (p < 0.05 for both comparisons). However, no statistically significant differences were observed between adenoma and papillary carcinoma tissues regarding BMI-1 gene expression. This study demonstrated a new biomarker for thyroid malignancies and found that the mRNA levels of the BMI-1 gene were higher in tumor tissues compared with healthy tissues. Further studies are needed to evaluate the BMI1 gene expression in other thyroid cancers.

Plasma Levels of MicroRNA-146a-5p, MicroRNA-24-3p, and MicroRNA125a-5p as Potential Diagnostic Biomarkers for Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by inflammation of the articular tissue. This study aims to evaluate the expression of microRNA (miR)-146a-5p, miR-24-3p, and miR-125a-5p in the plasma of RA patients and compare them with those of healthy controls to obtain a specific expression profile for earlier diagnosis and assistance in treating patients. This study was performed on 50 RA patients and 50 healthy controls. Five microliters of blood were taken from each patient/control. Plasma RNA was extracted using the Trisol solution. cDNAs were synthesized; using moloney murine leukemia virus (MMLV) and deoxynucleoside triphosphate (dNTP). Real-time PCR was performed using SYBR green kit. The mean expression of miR-146a-5p, miR-24-3p, and miR-125a-5p in the RA group were 8.1±1.9, 6.5±1.2, and 6.8±2.2 and in the healthy group were 4.8±1.6, 3.6±2.2, and 3.4±1.7, respectively. Significant differences were also observed in the mean expression of these three miRNAs in four subgroups of RA patients with different disease activity based on disease activity score 28 (DAS28) (p<0.05). ROC curve analysis showed that miR-146a-5p (AUC=0.8, sensitivity=96%, specificity=86%), miR-24-3p (AUC=0.7, Sensitivity=95%, Specificity=75%) and miR-125a-5p (AUC=0.71, sensitivity=93%, specificity=84%) could be used as suitable biomarkers for RA diagnosis. Increased expressions of miR-146a-5p, miR-24-3p, and miR-125a-5p in RA patients indicate that the miRNAs are involved in disease incidence and progression, and the measurement of their expression can play an essential role in the diagnosis and treatment of the disease.

Moloney murine leukemia virus p12 is required for histone loading onto retroviral DNAs

During retrovirus infection, a histone-free DNA copy of the viral RNA genome is synthesized and rapidly loaded with nucleosomes de novo upon nuclear entry. The potential role of viral accessory proteins in histone loading onto retroviral DNAs has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein critical for tethering the incoming viral DNA to host chromatin in the early stages of infection. Infection by virions containing a mutant p12 (PM14) defective in chromatin tethering results in the formation of viral DNAs that do not accumulate in the nucleus. In this report, we show that viral DNAs of these mutants are not loaded with histones. Moreover, the DNA genomes delivered by mutant p12 show prolonged association with viral structural proteins nucleocapsid (NC) and capsid (CA). The histone-poor viral DNA genomes do not become associated with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, indicating that tethering to host chromatin by p12 and retention in the nucleus are required to allow loading of histones onto the viral DNA. Importance: Incoming retroviral DNAs are rapidly loaded with nucleosomal histones upon entry into the nucleus and before integration into the host genome. The entry of murine leukemia virus DNA into the nucleus only occurs upon dissolution of the nuclear membrane in mitosis, and retention in the nucleus requires the action of a viral protein, p12, which tethers the DNA to host chromatin. Data presented here show that the tethering activity of p12 is required for the loading of histones onto the viral DNA. p12 mutants lacking tethering activity fail to acquire histones, retain capsid and nucleocapsid proteins, and are poorly transcribed. The work defines a new requirement for a viral protein to allow chromatinization of viral DNA.

PIM kinases mediate resistance to cisplatin chemotherapy in hepatoblastoma

Despite increasing incidence, treatment for hepatoblastoma has not changed significantly over the past 20 years. Chemotherapeutic strategies continue to rely on cisplatin, as it remains the most active single agent against hepatoblastoma. However, chemoresistance remains a significant challenge with 54–80% of patients developing resistance to chemotherapy after 4–5 cycles of treatment. Stem cell-like cancer cells (SCLCCs) are a subset of cells thought to play a role in chemoresistance and disease recurrence. We have previously demonstrated that Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, specifically PIM3, play a role in hepatoblastoma cell proliferation and tumor growth and maintain the SCLCC phenotype. Here, we describe the development of a cisplatin-resistant hepatoblastoma xenograft model of the human HuH6 cell line and a patient-derived xenograft, COA67. We provide evidence that these cisplatin-resistant cells are enriched for SCLCCs and express PIM3 at higher levels than cisplatin-naïve cells. We demonstrate that PIM inhibition with AZD1208 sensitizes cisplatin-resistant hepatoblastoma cells to cisplatin, enhances cisplatin-mediated apoptosis, and decreases the SCLCC phenotype seen with cisplatin resistance. Together, these findings indicate that PIM inhibition may be a promising adjunct in the treatment of hepatoblastoma to effectively target SCLCCs and potentially decrease chemoresistance and subsequent disease relapse.

Improvement of Moloney murine leukemia virus reverse transcriptase thermostability by introducing a disulfide bridge in the ribonuclease H region

Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) is widely used in research and clinical diagnosis. Improvement of MMLV RT thermostability has been an important topic of research for increasing the efficiency of cDNA synthesis. In this study, we attempted to increase MMLV RT thermostability by introducing a disulfide bridge in its RNase H region using site-directed mutagenesis. Five variants were designed, focusing on the distance between the two residues to be mutated into cysteine. The variants were expressed in Escherichia coli and purified. A551C/T662C was determined to be the most thermostable variant.

Efficient production of Moloney murine leukemia virus-like particles pseudotyped with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein

ABSTRACTThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak that started in China at the end of 2019 has rapidly spread to become pandemic. Several investigational vaccines that have already been tested in animals and humans were able to induce neutralizing antibodies against the SARS-CoV-2 spike (S) protein, however protection and long-term efficacy in humans remain to be demonstrated.We have investigated if a virus-like particle (VLP) derived from Moloney murine leukemia virus (MLV) could be engineered to become a candidate SARS-CoV-2 vaccine amenable to mass production. First, we showed that a codon optimized version of the S protein could migrate efficiently to the cell membrane. However, efficient production of infectious viral particles was only achieved with stable expression of a shorter version of S in its C-terminal domain (ΔS) in 293 cells that express MLV Gag-Pol (293GP). The incorporation of ΔS was 15-times more efficient into VLPs as compared to the full-length version, and that was not due to steric interference between the S cytoplasmic tail and the MLV capsid. Indeed, a similar result was also observed with extracellular vesicles released from parental 293 and 293GP cells. The amount of ΔS incorporated into VLPs released from producer cells was robust, with an estimated 1.25 μg/ml S2 equivalent (S is comprised of S1 and S2). Thus, a scalable platform that has the potential for production of pan-coronavirus VLP vaccines has been established. The resulting nanoparticles could potentially be used alone or as a boost for other immunization strategies for COVID-19.IMPORTANCESeveral candidate COVID-19 vaccines have already been tested in humans, but their protective effect and long-term efficacy are uncertain. Therefore, it is necessary to continue developing new vaccine strategies that could be more potent and/or that would be easier to manufacture in large-scale. Virus-like particle (VLP) vaccines are considered highly immunogenic and have been successfully developed for human papilloma virus as well as hepatitis and influenza viruses. In this study, we report the generation of a robust Moloney murine leukemia virus platform that produces VLPs containing the spike of SARS-CoV-2. This vaccine platform that is compatible with lyophilization could simplify storage and distribution logistics immensely.

R3T (Rapid Research Response Team) One-step RT-qPCR kit for COVID-19 diagnostic using in-house enzymes

One-step RT-qPCR is the most widely applied method for COVID-19 diagnostics. Designing in-house one-step RT-qPCR kits is restricted by the patent-rights for the production of enzymes and the lack of information about the components of the commercial kits. Here, we provide a simple, economical, and powerful one-step RT-qPCR kit based on patent-free, specifically-tailored versions of Moloney Murine Leukemia Virus Reverse Transcriptase and Thermus aquaticus DNA polymerase termed the R3T (Rapid Research Response Team) One-step RT-qPCR. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of the SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity as that of the Invitrogen SuperScript III Platinum One-step RT-qPCR and TaqPath 1-Step RT-qPCR kits. Overall, our kit has shown robust performance in both of laboratory settings and the Saudi Ministry of Health-approved testing facility.

Screening and Evaluation of Novel Compounds against Hepatitis B Virus Polymerase Using Highly Purified Reverse Transcriptase Domain

Hepatitis B virus (HBV) polymerase seems to be very hard to express and purify sufficiently, which has long hampered the generation of anti-HBV drugs based on the nature of the polymerase. To date, there has been no useful system developed for drug screening against HBV polymerase. In this study, we successfully obtained a highly purified reverse transcriptase (RT) domain of the polymerase, which has a template/primer and substrate binding activity, and established a novel high-throughput screening (HTS) system using purified RT protein for finding novel polymerase inhibitors. To examine whether the assay system provides reliable results, we tested the small scale screening using pharmacologically active compounds. As a result, the pilot screening identified already-known anti-viral polymerase agents. Then, we screened 20,000 chemical compounds and newly identified four hits. Several of these compounds inhibited not only the HBV RT substrate and/ template/primer binding activity, but also Moloney murine leukemia virus RT activity, which has an elongation activity. Finally, these candidates did show to be effective even in the cell-based assay. Our screening system provides a useful tool for searching candidate inhibitors against HBV.