Chemotherapeutic Agents from Natural Product Templates: The Design and Synthesis of Novel Indanone Analogues

Coumarin is a natural product found in many plants. Recently, simple coumarin analogues containing an α-methylene functional group have been synthesized and shown to exhibit cytotoxicity against cancer cell lines. The α,β-unsaturated carbonyl found in these analogues is thought to be responsible for their bioactivity. Other natural products containing this functional group have been shown to react with intracellular thiols causing tumor growth suppression. Using these analogues as a template, two new compounds have been designed that feature a novel α-methylene indanone structural framework. Since ketones are better electrophiles that esters, it is hypothesized that these analogues will react more rapidly with thiols that their coumarin counterparts. The two α-methylene indanone analogues, one incorporating an isopropyl and the other an n-butyl substituent, have been prepared in five and seven steps respectively. Both synthetic sequences feature a 1,4-organometallic addition reaction, in intramolecular Friedel-Crafts acylation, and an α-methylenation of the key inadone intermediates

NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2

Background: The capacity of patient's Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling's NKs were compared with the patient's drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient's autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared. Methods: Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling's NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody Rituximab (TM) were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays. Results: Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient's autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission. Conclusions: Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML

ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5' tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding

ATP hydrolysis by the viral RNA sensor RIG-I prevents unintentional recognition of self-RNA

The cytosolic antiviral innate immune sensor RIG-I distinguishes 5' tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding

The USP7/Dnmt1 complex stimulates the DNA methylation activity of Dnmt1 and regulates the stability of UHRF1

Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo

Multi-ancestry genome-wide association meta-analysis of Parkinson?s disease

Although over 90 independent risk variants have been identified for Parkinson’s disease using genome-wide association studies, most studies have been performed in just one population at a time. Here we performed a large-scale multi-ancestry meta-analysis of Parkinson’s disease with 49,049 cases, 18,785 proxy cases and 2,458,063 controls including individuals of European, East Asian, Latin American and African ancestry. In a meta-analysis, we identified 78 independent genome-wide significant loci, including 12 potentially novel loci (MTF2, PIK3CA, ADD1, SYBU, IRS2, USP8, PIGL, FASN, MYLK2, USP25, EP300 and PPP6R2) and fine-mapped 6 putative causal variants at 6 known PD loci. By combining our results with publicly available eQTL data, we identified 25 putative risk genes in these novel loci whose expression is associated with PD risk. This work lays the groundwork for future efforts aimed at identifying PD loci in non-European populations

Defining the causes of sporadic Parkinson's disease in the global Parkinson's genetics program (GP2)

The Global Parkinson’s Genetics Program (GP2) will genotype over 150,000 participants from around the world, and integrate genetic and clinical data for use in large-scale analyses to dramatically expand our understanding of the genetic architecture of PD. This report details the workflow for cohort integration into the complex arm of GP2, and together with our outline of the monogenic hub in a companion paper, provides a generalizable blueprint for establishing large scale collaborative research consortia

Syntheses and Structure–Activity Relationships of N-Phenethyl-Quinazolin-4-yl-Amines as Potent Inhibitors of Cytochrome bd Oxidase in Mycobacterium tuberculosis

The development of cytochrome bd oxidase (cyt-bd) inhibitors are needed for comprehensive termination of energy production in Mycobacterium tuberculosis (Mtb) to treat tuberculosis infections. Herein, we report on the structure-activity-relationships (SAR) of 22 new N-phenethyl-quinazolin-4-yl-amines that target cyt-bd. Our focused set of compounds was synthesized and screened against three mycobacterial strains: Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and the clinical isolate Mycobacterium tuberculosis N0145 with and without the cytochrome bcc:aa3 inhibitor Q203 in an ATP depletion assay. Two compounds, 12a and 19a, were more active against all three strains than the naturally derived cyt-bd inhibitor aurachin D

NK cells from an AML patient have recovered in remission and reached comparable cytolytic activity to that of a healthy monozygotic twin mediated by the single-chain triplebody SPM-2

Background The capacity of patient’s Natural Killer cells (NKs) to be activated for cytolysis is an important prerequisite for the success of antibody-derived agents such as single-chain triplebodies (triplebodies) in cancer therapy. NKs recovered from AML patients at diagnosis are often found to be reduced in peripheral blood titers and cytolytic activity. Here, we had the unique opportunity to compare blood titers and cytolytic function of NKs from an AML patient with those of a healthy monozygotic twin. The sibling’s NKs were compared with the patient’s drawn either at diagnosis or in remission after chemotherapy. The cytolytic activities of NKs from these different sources for the patient’s autologous AML blasts and other leukemic target cells in conjunction with triplebody SPM-2, targeting the surface antigens CD33 and CD123 on the AML cells, were compared. Methods Patient NKs drawn at diagnosis were compared to NKs drawn in remission after chemotherapy and a sibling’s NKs, all prepared from PBMCs by immunomagnetic beads (MACS). Redirected lysis (RDL) assays using SPM-2 and antibody-dependent cellular cytotoxicity (ADCC) assays using the therapeutic antibody RituximabTM were performed with the enriched NKs. In addition, MACS-sorted NKs were analyzed for NK cell activating receptors (NCRs) by flow cytometry, and the release of TNF-alpha and IFN-gamma from blood samples of both siblings after the addition of the triplebody were measured in ELISA-assays. Results Patient NKs isolated from peripheral blood drawn in remission produced comparable lysis as NKs from the healthy twin against the patient’s autologous bone marrow (BM) blasts, mediated by SPM-2. The NCR receptor expression profiles on NKs from patient and twin were similar, but NK cell titers in peripheral blood were lower for samples drawn at diagnosis than in remission. Conclusions Peripheral blood NK titers and ex vivo cytolytic activities mediated by triplebody SPM-2 were comparable for cells drawn from an AML patient in remission and a healthy twin. If these results can be generalized, then NKs from AML patients in remission are sufficient in numbers and cytolytic activity to make triplebodies promising new agents for the treatment of AML

In Vitro Characterization of Hypoxia Preconditioned Serum (HPS)—Fibrin Hydrogels: Basis for an Injectable Biomimetic Tissue Regeneration Therapy

Blood-derived growth factor preparations have long been employed to improve perfusion and aid tissue repair. Among these, platelet-rich plasma (PRP)-based therapies have seen the widest application, albeit with mixed clinical results to date. Hypoxia-preconditioned blood products present an alternative to PRP, by comprising the complete wound healing factor-cascade, i.e., hypoxia-induced peripheral blood cell signaling, in addition to platelet-derived factors. This study set out to characterize the preparation of hypoxia preconditioned serum (HPS), and assess the utility of HPS–fibrin hydrogels as vehicles for controlled factor delivery. Our findings demonstrate the positive influence of hypoxic incubation on HPS angiogenic potential, and the individual variability of HPS angiogenic factor concentration. HPS–fibrin hydrogels can rapidly retain HPS factor proteins and gradually release them over time, while both functions appear to depend on the fibrin matrix mass. This offers a means of controlling factor retention/release, through adjustment of HPS fibrinogen concentration, thus allowing modulation of cellular angiogenic responses in a growth factor dose-dependent manner. This study provides the first evidence that HPS–fibrin hydrogels could constitute a new generation of autologous/bioactive injectable compositions that provide biochemical and biomaterial signals analogous to those mediating physiological wound healing. This therefore establishes a rational foundation for their application towards biomimetic tissue regeneration