Identification of Anti-tubercular Compounds in Marine Organisms from Aotearoa

Tuberculosis (TB) is responsible for more than one million human deaths per year globally, more than any other disease caused by a bacterial pathogen. Although effective treatments exist, the long duration of the current treatment regimen as well as associated drug toxicities can lead to patient non-compliance. This has resulted in the spread of drug resistance which is highlighted by the isolation of extensively drug-resistant strains of Mycobacterium tuberculosis complex in all regions of the globe. The management of drug-resistant TB cases requires an extended treatment duration of 18 months minimum post sputum-culture conversion, using drugs that are more frequently linked to adverse side-effects. Therefore, there is a need to identify new drugs that shorten treatment time, and also exhibit better tolerance in patients. This thesis describes the screening of crude marine extract libraries for the identification of novel anti-tubercular compounds. The libraries consisted of extracts from New Zealand marine organisms that were screened for growth inhibitory activity using a surrogate TB model organism M. smegmatis, and subsequently were validated against M. tuberculosis. Bioassay-guided fractionation led to the identification of two new compounds as well as a mixture of known compounds. In addition to anti-mycobacterial activity, the spectrum of activity of the isolated compounds was also investigated. Fractionation of the liver extract of the blobfish Psychrolutes marcidus led to the identification of a mixture of four common fatty acids as the major bioactive components. Testing each of the fatty acids individually identified palmitic acid as the most active component against M. tuberculosis H37Ra. This is at odds with current understanding of the fatty acid parameters required for bioactivity in terms of chain length and degree of unsaturation. Bioassay-guided fractionation of a Latrunculia sp. sponge extract led to the isolation of a novel taurinated terpene rimarikiamide A (98). This compound was initially believed to be responsible for the observed anti-mycobacterial activity, however, further purification led to partitioning of the anti-mycobacterial activity away from 98. The taurinated terpene rimarikiamide A was nevertheless tested for activity in mammalian cell lines and was found to exhibit higher levels of growth inhibition towards HL-60 cells with respect to HEK cells. Working with Xiphophora chondrophylla, a New Zealand brown alga, led to the identification of the amino alcohol 3-epi-xestoaminol C (156) a stereoisomer of a known compound, as the active principle. This compound displayed the same level of activity in all microorganisms tested, however it proved to be more active against HL-60 cells. To further investigate the mode of action of 156, chemical genetics was employed to indicate the relevant genetic networks that the compound interacts with. This led to the identification of expected pathways such as lipid metabolism as well as cytoskeletal organization, which have been linked to this class of compounds. Furthermore this work also identified new processes such as RNA catabolism, protein targeting and phosphorylation in addition to several unknown processes

Identification of Anti-tubercular Compounds in Marine Organisms from Aotearoa

Tuberculosis (TB) is responsible for more than one million human deaths per year globally, more than any other disease caused by a bacterial pathogen. Although effective treatments exist, the long duration of the current treatment regimen as well as associated drug toxicities can lead to patient non-compliance. This has resulted in the spread of drug resistance which is highlighted by the isolation of extensively drug-resistant strains of Mycobacterium tuberculosis complex in all regions of the globe. The management of drug-resistant TB cases requires an extended treatment duration of 18 months minimum post sputum-culture conversion, using drugs that are more frequently linked to adverse side-effects. Therefore, there is a need to identify new drugs that shorten treatment time, and also exhibit better tolerance in patients. This thesis describes the screening of crude marine extract libraries for the identification of novel anti-tubercular compounds. The libraries consisted of extracts from New Zealand marine organisms that were screened for growth inhibitory activity using a surrogate TB model organism M. smegmatis, and subsequently were validated against M. tuberculosis. Bioassay-guided fractionation led to the identification of two new compounds as well as a mixture of known compounds. In addition to anti-mycobacterial activity, the spectrum of activity of the isolated compounds was also investigated. Fractionation of the liver extract of the blobfish Psychrolutes marcidus led to the identification of a mixture of four common fatty acids as the major bioactive components. Testing each of the fatty acids individually identified palmitic acid as the most active component against M. tuberculosis H37Ra. This is at odds with current understanding of the fatty acid parameters required for bioactivity in terms of chain length and degree of unsaturation. Bioassay-guided fractionation of a Latrunculia sp. sponge extract led to the isolation of a novel taurinated terpene rimarikiamide A (98). This compound was initially believed to be responsible for the observed anti-mycobacterial activity, however, further purification led to partitioning of the anti-mycobacterial activity away from 98. The taurinated terpene rimarikiamide A was nevertheless tested for activity in mammalian cell lines and was found to exhibit higher levels of growth inhibition towards HL-60 cells with respect to HEK cells. Working with Xiphophora chondrophylla, a New Zealand brown alga, led to the identification of the amino alcohol 3-epi-xestoaminol C (156) a stereoisomer of a known compound, as the active principle. This compound displayed the same level of activity in all microorganisms tested, however it proved to be more active against HL-60 cells. To further investigate the mode of action of 156, chemical genetics was employed to indicate the relevant genetic networks that the compound interacts with. This led to the identification of expected pathways such as lipid metabolism as well as cytoskeletal organization, which have been linked to this class of compounds. Furthermore this work also identified new processes such as RNA catabolism, protein targeting and phosphorylation in addition to several unknown processes

Identification of Anti-tubercular Compounds in Marine Organisms from Aotearoa

Tuberculosis (TB) is responsible for more than one million human deaths per year globally, more than any other disease caused by a bacterial pathogen. Although effective treatments exist, the long duration of the current treatment regimen as well as associated drug toxicities can lead to patient non-compliance. This has resulted in the spread of drug resistance which is highlighted by the isolation of extensively drug-resistant strains of Mycobacterium tuberculosis complex in all regions of the globe. The management of drug-resistant TB cases requires an extended treatment duration of 18 months minimum post sputum-culture conversion, using drugs that are more frequently linked to adverse side-effects. Therefore, there is a need to identify new drugs that shorten treatment time, and also exhibit better tolerance in patients.  This thesis describes the screening of crude marine extract libraries for the identification of novel anti-tubercular compounds. The libraries consisted of extracts from New Zealand marine organisms that were screened for growth inhibitory activity using a surrogate TB model organism M. smegmatis, and subsequently were validated against M. tuberculosis. Bioassay-guided fractionation led to the identification of two new compounds as well as a mixture of known compounds. In addition to anti-mycobacterial activity, the spectrum of activity of the isolated compounds was also investigated.  Fractionation of the liver extract of the blobfish Psychrolutes marcidus led to the identification of a mixture of four common fatty acids as the major bioactive components. Testing each of the fatty acids individually identified palmitic acid as the most active component against M. tuberculosis H37Ra. This is at odds with current understanding of the fatty acid parameters required for bioactivity in terms of chain length and degree of unsaturation.  Bioassay-guided fractionation of a Latrunculia sp. sponge extract led to the isolation of a novel taurinated terpene rimarikiamide A (98). This compound was initially believed to be responsible for the observed anti-mycobacterial activity, however, further purification led to partitioning of the anti-mycobacterial activity away from 98. The taurinated terpene rimarikiamide A was nevertheless tested for activity in mammalian cell lines and was found to exhibit higher levels of growth inhibition towards HL-60 cells with respect to HEK cells.  Working with Xiphophora chondrophylla, a New Zealand brown alga, led to the identification of the amino alcohol 3-epi-xestoaminol C (156) a stereoisomer of a known compound, as the active principle. This compound displayed the same level of activity in all microorganisms tested, however it proved to be more active against HL-60 cells. To further investigate the mode of action of 156, chemical genetics was employed to indicate the relevant genetic networks that the compound interacts with. This led to the identification of expected pathways such as lipid metabolism as well as cytoskeletal organization, which have been linked to this class of compounds. Furthermore this work also identified new processes such as RNA catabolism, protein targeting and phosphorylation in addition to several unknown processes.</p

Selecting costimulatory domains for chimeric antigen receptors: functional and clinical considerations

Abstract Costimulatory signals are required to achieve robust chimeric antigen receptor (CAR) T cell expansion, function, persistence and antitumor activity. These can be provided by incorporating intracellular signalling domains from one or more T cell costimulatory molecules, such as CD28 or 4‐1BB, into the CAR. The selection and positioning of costimulatory domains within a CAR construct influence CAR T cell function and fate, and clinical experience of autologous anti‐CD19 CAR T cell therapies suggests that costimulatory domains have differential impacts on CAR T cell kinetics, cytotoxic function and potentially safety profile. The clinical impacts of combining costimulatory domains and of alternative costimulatory domains are not yet clearly established, and may be construct‐ and disease‐specific. The aim of this review is to summarise the function and effect of established and emerging costimulatory domains and their combinations within CAR T cells

Identification and Bioactivity of 3-<i>epi</i>-Xestoaminol C Isolated from the New Zealand Brown Alga <i>Xiphophora chondrophylla</i>

We report here the bioassay-guided isolation of a new 1-deoxysphingoid, 3-<i>epi</i>-xestoaminol C (<b>1</b>), isolated from the New Zealand brown alga <i>Xiphophora chondrophylla</i>. This is the first report of a 1-deoxysphingoid from a brown alga. We describe the isolation and full structure elucidation of this compound, including its absolute configuration, along with its bioactivity against mycobacteria and mammalian cell lines and preliminary mechanism of action studies using yeast chemical genomics

Third-generation anti-CD19 chimeric antigen receptor T-cells incorporating a TLR2 domain for relapsed or refractory B-cell lymphoma: a phase I clinical trial protocol (ENABLE).

INTRODUCTION: Autologous T-cells transduced to express a chimeric antigen receptor (CAR) directed against CD19 elicit high response rates in relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (B-NHL). However, r/r B-NHL remissions are durable in fewer than half of recipients of second-generation CAR T-cells. Third-generation (3G) CARs employ two costimulatory domains, resulting in improved CAR T-cell efficacy in vitro and in animal models in vivo. This investigator-initiated, phase I dose escalation trial, termed ENABLE, will investigate the safety and preliminary efficacy of WZTL-002, comprising autologous T-cells expressing a 3G anti-CD19 CAR incorporating the intracellular signalling domains of CD28 and Toll-like receptor 2 (TLR2) for the treatment of r/r B-NHL. METHODS AND ANALYSIS: Eligible participants will be adults with r/r B-NHL including diffuse large B-cell lymphoma and its variants, follicular lymphoma, transformed follicular lymphoma and mantle cell lymphoma. Participants must have satisfactory organ function, and lack other curative options. Autologous T-cells will be obtained by leukapheresis. Following WZTL-002 manufacture and product release, participants will receive lymphodepleting chemotherapy comprising intravenous fludarabine and cyclophosphamide. A single dose of WZTL-002 will be administered intravenously 2 days later. Targeted assessments for cytokine release syndrome and immune cell effector-associated neurotoxicity syndrome, graded by the American Society Transplantation and Cellular Therapy criteria, will be made. A modified 3+3 dose escalation scheme is planned starting at 5×104 CAR T-cells/kg with a maximum dose of 1×106 CAR T-cells/kg. The primary outcome of this trial is safety of WZTL-002. Secondary outcomes include feasibility of WZTL-002 manufacture and preliminary measures of efficacy. ETHICS AND DISSEMINATION: Ethical approval for the study was granted by the New Zealand Health and Disability Ethics Committee (reference 19/STH/69) on 23 June 2019 for Protocol V.1.2. Trial results will be reported in a peer-reviewed journal, and results presented at scientific conferences or meetings. TRIAL REGISTRATION NUMBER: NCT04049513

Intratumoural administration of an NKT cell agonist with CpG promotes NKT cell infiltration associated with an enhanced antitumour response and abscopal effect

Intratumoural administration of unmethylated cytosine-phosphate-guanine motifs (CpG) to stimulate toll-like receptor (TLR)-9 has been shown to induce tumour regression in preclinical studies and some efficacy in the clinic. Because activated natural killer T (NKT) cells can cooperate with pattern-recognition via TLRs to improve adaptive immune responses, we assessed the impact of combining a repeated dosing regimen of intratumoural CpG with a single intratumoural dose of the NKT cell agonist α-galactosylceramide (α-GalCer). The combination was superior to CpG alone at inducing regression of established tumours in several murine tumour models, primarily mediated by CD8+ T cells. An antitumour effect on distant untreated tumours (abscopal effect) was reliant on sustained activity of NKT cells and was associated with infiltration of KLRG1+ NKT cells in tumours and draining lymph nodes at both injected and untreated distant sites. Cytometric analysis pointed to increased exposure to type I interferon (IFN) affecting many immune cell types in the tumour and lymphoid organs. Accordingly, antitumour activity was lost in animals in which dendritic cells (DCs) were incapable of signaling through the type I IFN receptor. Studies in conditional ablation models showed that conventional type 1 DCs and plasmacytoid DCs were required for the response. In tumour models where the combined treatment was less effective, the addition of tumour-antigen derived peptide, preferably conjugated to α-GalCer, significantly enhanced the antitumour response. The combination of TLR ligation, NKT cell agonism, and peptide delivery could therefore be adapted to induce responses to both known and unknown antigens