Identification of novel Arf1 GTPase inhibitors for cancer target validation
- Authors: Mqwathi, Nomxolisi Vuyokasi
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Guanosine triphosphatase , Cancer Treatment
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424666 , vital:72173
- Description: The key regulators of both anterograde and retrograde vesicular traffic, adenosine diphosphate-ribosylation factors (Arfs), also coordinate various signalling pathways and regulate cellular processes required for cell survival and function. In addition to its role in mediating secretory trafficking in the Golgi apparatus, the involvement of Arf1 in signalling pathways that contribute to the formation and progression of cancer has become apparent, and the overexpression and deregulation of Arf1 activity has been associated with cancer cell invasion, proliferation and metastasis. As with other small GTPases, Arf1 must cycle back and forth between an inactive (GDP-bound) and active (GTP-bound) conformation to carry out its function. However, the cycle of Arf1 inactivation and activation is controlled by Arf GTPase activating proteins (Arf-GAPs) that stimulate Arf1 to hydrolyse the bound GTP to GDP and Arf guanine nucleotide exchange factors (Arf-GEFs) that facilitate GDP for GTP exchange on Arf1, respectively. The identification of Arf1 inhibitors that indirectly disrupt Arf1 function by blocking its interaction with Arf-GAPs or Arf-GEFs has generated interest in their use as possible anti-cancer agents. The suppression of Arf1 activation (by targeting Arf-GEFs) has been investigated as a potential cancer therapeutic target and resulted in inhibitor compounds that have micromolar-range activity against cancer cells and targets and promising results in mouse models, but experience problems with bioavailability when used in vivo. This motivates the search for novel Arf1 inhibitors for validation purposes to question whether Arf1 is a viable target for cancer therapy. The purpose of the study was to employ a recently developed colourimetric screening assay to identify inhibitors of Arf1 activation (Arf-GEF inhibitors) and deactivation (Arf-GAP inhibitors), with a focus on evaluating the potential of Arf1 deactivation as an entirely novel anti-cancer target. The proteins required for the assay (Arf1, Arf-GEF and -GAP domains and a reporter protein, GST-GGA3) were expressed in E. coli. and purified using affinity chromatography. The assay could detect the activation of Arf1 by the catalytic Sec7 domain of the three Arf-GEFs chosen for this study, but reproducibility was compromised by the occasional spontaneous activation of Arf1 in the absence of the Arf-GEFs. By contrast, the assay could reproducibly detect Arf1 deactivation by an Arf-GAP domain (Arf-GAP1GAP) and was subsequently used to screen a library of α-helix mimetics. Thirteen hit compounds with IC50 values ranging from 0.53 to 20.95 μM were found to inhibit Arf-GAP1GAP-mediated stimulation of GTP hydrolysis by Arf1-GTP in this assay format, however, they did not effectively suppress the proliferation of three tested cell lines (HeLa, MCF-7 and MCF-12A). Interestingly, the results obtained from fluorescence microscopy studies suggested that the compounds disrupt Golgi structure and Arf1 localisation, presumably by keeping Arf1 in its active conformation by blocking Arf-GAP1 function. This suggests that the compounds affect Arf1 function in cells, and may be used to explore the feasibility of targeting Arf1 deactivation for anti-cancer purposes in a wider range of cell lines and experiments. It has been reported that Arf-GAP1 inhibition is associated with the suppression of cell migration, and the potential of the compounds as metastasis inhibitors may also be explored. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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- Date Issued: 2023-10-13
The development of a plate-based assay to detect the activation status of ARF1 GTPase in Plasmodium falciparum parasites
- Authors: Du Toit, Skye Carol
- Date: 2023-10-13
- Subjects: ARF1 , GTPase , Plasmodium falciparum , Malaria , Drug resistance , Drug targeting , Enzyme-linked immunosorbent assay , Proteins
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424654 , vital:72172
- Description: The exponential rise in antimalarial drug resistance in the most infectious malaria species, Plasmodium falciparum, has emphasised the urgency to identify and validate novel drug targets that decrease parasite viability upon inhibition. In addition to several publications indicating that the regulation of human Arf1 GTPase activity (mediated by ArfGEFs and ArfGAPs) serves as a pertinent drug target for cancer research, the identification of Arf1 and its regulatory proteins in Plasmodium falciparum led to the question whether these protein homologs could be exploited as drug targets for anti-malarial drug therapies. To investigate this prospect, the establishment of a novel in vitro colorimetric ELISA-based assay was needed to be able to detect changes in the activation status of P. falciparum Arf1 (PfArf1) in parasite cultures exposed to potential Arf1 inhibitors. By exploiting the selective protein interaction that occurs between active GTP-bound Arf1 and its downstream effector, GGA3, an assay protocol was established that could be used to detect the activation status of purified, truncated PfArf1 obtained from E. coli and endogenous PfArf1 sourced from parasite lysates. The assay relies on the use of anti-Arf1 antibodies to detect the binding of active PfArf1 in the lysates of inhibitor-exposed cultured parasites to GST-GGA3 immobilised in glutathione-coated plates. The results from chemical validation experiments conducted using the novel assay developed in this study, using the known ArfGEF inhibitor brefeldin A (BFA) and ArfGAP inhibitors Chem1099 and Chem3050, yielded the anticipated results: decrease in active PfArf1 after parasite incubation with the ArfGEF inhibitor, and increased active PfArf1 after ArfGAP inhibition. The results confirmed PfArf1 as a potential anti-malarial drug target and encourages the further development of this assay format for the identification of subsequent inhibitors in library screening campaigns. Additional pilot experiments were conducted to further explore whether the assay could detect the activation status of human Arf1 using HeLa cell lysates and to provide further evidence that the assay could be exploited as a tool in the identification of Arf1 GTPase inhibitors with BFA and the known ArfGAP inhibitor, QS11. The results suggested that, while the assay can detect the increase in active cellular Arf1 due to the inhibition of human ArfGEF following BFA treatment, subsequent treatment with QS11 showed no evidence of a reduction in active human Arf1 due to ArfGAP inhibition. Further experimentation is required to investigate the ability the assay to confirm inhibition of human Arf1 deactivation by ArfGAP inhibitors and develop the assay as a useful tool to support cancer drug discovery, in addition to antimalarial drug discovery projects aimed at Arf1. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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- Date Issued: 2023-10-13
Exploring the use of in vitro colorimetric and bioluminescence assays to distinguish between Arf GTPase isoforms and detect Arf GTPase activity
- Authors: Woolf, Alexander Robert
- Date: 2021-10-29
- Subjects: ADP ribosylation factor , ARF1 , ARF6 , ARFGAP1 , GGA3 , NDPK
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192582 , vital:45240
- Description: ADP-ribosylation factors (Arfs), part of the Ras superfamily of small GTPases, are involved in various key cellular processes such as the regulation of membrane trafficking and organelle structure. Arfs cycle between a GDP-bound ‘inactive’ conformation and a GTP-bound ‘active’ conformation, with the latter exerting its functions on various downstream effector proteins. In eukaryotic organisms, Arf6 regulates traffic in the endocytic pathway and contributes the reorganisation of actin cytoskeleton. Due to its ability to affect health and disease in many ways, Arf6 has been a viable drug target for many diseases, with this study focusing mainly on cancer and malaria. In the P. falciparum parasite genome are two genes encoding for Arf GTPase. The first (PfArf1) has been designated as an Arf1 homologue, and the second shares sequence similarities with both human Arf1 and Arf6 but is otherwise uncharacterised. The potential of the second putative malarial Arf sequence to be a homologue of human Arf6 is important as it would further our understanding of the unusual mechanisms behind malarial endocytosis of the red blood cell cytoplasm. To assign this second putative malarial Arf (PfArf6) as a human Arf6 or Arf1 homologue, a novel plate-based colorimetric assay system was used involving the Arf effector binding domains of “golgi-localised gamma adaptin ear-containing ARF-binding protein 3” (GGA3), which interacts with Arf1 and Arf6, and “c-Jun NH2-terminal kinase-interacting protein 4” (JIP4), which interacts with Arf6 only. It was shown that PfArf6 was able to bind to both GGA3 and JIP4 and that PfArf6 was distinguishable from PfArf1. Initial data obtained designated “PfArf6” as an Arf6 homologue, despite the higher sequence similarity between PfArf6 and human Arf1. Arf GTPases display notoriously slow intrinsic GTPase activity and so their activity is tightly regulated by their cognate guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs), which cycle Arfs between their GDP- and GTP-bound conformations. Arf6 and its closely associated regulators are involved extensively in the promotion of tumour growth, invasion, and metastasis stages of cancer. Simple and reproducible GTPase activity assays, that are simultaneously amenable to screening compound libraries for Arf inhibitors, are limited in availability due to the difficulties surrounding accurate GTP detection. A novel in vitro plate-based bioluminescence assay format developed in this study to detect Arf GTPase activity in the presence of its regulators namely “ARF nucleotide-binding site opener” (ARNO) and ArfGAP1. In cells “nucleoside diphosphate kinase” (NDPK) functions to regulate the available nucleoside triphosphate (NTP) pools to maintain homeostasis. Given the relative ease for accurate ATP detection using firefly luciferase and the ability for NDPK the convert GTP to ATP, NDPK and luciferase were utilised in this coupled assay to detect GTP consumption by Arf. Regulator stimulated Arf GTPase activity was eventually detectable to varying degrees for human Arf1 and Arf6, as well as malarial Arf1. Thus, the groundwork for a simple, plate-based assay system has been laid for the detection of the full Arf GTPase activity cycle, the screening of compound libraries for Arf inhibitors, as well as a means of determining Arf GTPase specificity for regulators (and vice versa) in vitro. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
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- Date Issued: 2021-10-29