Postgraduate Thesis: Mechanism study of a small molecule F18 as a novel anti-HIV-1 non-nucleoside reverse transcriptase inhibitor

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Title	Mechanism study of a small molecule F18 as a novel anti-HIV-1 non-nucleoside reverse transcriptase inhibitor
Authors	Lu, Xiaofan. 陆小凡.
Issue Date	2012
Publisher	The University of Hong Kong (Pokfulam, Hong Kong)
Abstract	Non-nucleoside reverse transcriptase inhibitor (NNRTI) is one of the key components of antiretroviral drug regimen against human immunodeficiency virus type-1 (HIV-1) replication. However, the low genetic barriers to drug-resistance or cross-resistance, side effects, as well as the unaffordable cost of NNRTIs compromise their clinical usage. Therefore, to develop novel NNRTIs with potent antiviral activity against HIV-1 becomes a major concern in the treatment and prevention of HIV/AIDS. (+)-Calanolide A, which is a natural product initially extracted from the tropical rainforest tree Calophyllum lanigerum, was identified as an attractive NNRTI against HIV-1 despite virus strains containing drug-resistant K103N/Y181C mutations. In this study, a chemical library was constructed based on the three chiral carbon centers of (+)-Calanolide A. After screening the activity against HIVNL4-3 wild-type and several NNRTI-resistant pseudoviruses, a small molecule 10-chloromethyl-11- demethyl-12-oxo-calanolide A (F18) was identified as novel NNRTI with promising anti-HIV efficacy. Further studies were performed to investigate the antiviral breadth, drug resistance profile and underlying mechanism of the action of F18. F18 consistently displayed a potent activity against primary HIV-1 isolates including various subtypes of M group, CRF01_AE, and laboratory-adapted drug-resistant viruses in PBMC based assay. Moreover, F18 displayed distinct profiles against 17 NNRTI-resistant pseudoviruses, with an excellent potency especially against one of the most prevalent strains with the Y181C mutation (EC50=1.0nM) in cell line based assay, which was in stark contrast from the extensively used NNRTIs nevirapine and efavirenz. F18-resistant viruses were induced by in vitro serial passages, and mutation L100I was appeared to be the dominant contributor to F18-resistance, further suggesting a binding motif different from nevirapine and efavirenz. The efficacy of F18 was non-antagonistic when used in combination with other antiretrovirals against both wild-type and drug-resistant viruses in infected PBMCs. Interestingly, F18 displayed a highly synergistic antiviral effect with nevirapine against nevirapine-resistant virus (Y181C). Furthermore, in silico docking analysis suggested that F18 may bind to the HIV-1 reverse transcriptase in a way different to other NNRTIs. For the potential as an anti-HIV-1 microbicide, F18 also showed the stable and rapid release, as well as the sustained antiviral activity against HIV-1 wild-type virus in a formulation temperature-sensitive acidic gel. In summary, this study presents F18 as a new potential drug for clinical use and also underlies new mechanism-based design for future NNRTI.
Advisors	Chen, Z Zheng, B
Degree	Doctor of Philosophy
Subject	AIDS (Disease) - Chemotherapy. HIV infections - Chemotherapy. Reverse transcriptase - Inhibitors - Therapeutic use. Antiretroviral agents.
Dept/Program	Microbiology
DOI	http://dx.doi.org/10.5353/th_b4724650

DC Field	Value
dc.contributor.advisor	Chen, Z
dc.contributor.advisor	Zheng, B
dc.contributor.author	Lu, Xiaofan.
dc.contributor.author	陆小凡.
dc.date.hkucongregation	2012
dc.date.issued	2012
dc.description.abstract	Non-nucleoside reverse transcriptase inhibitor (NNRTI) is one of the key components of antiretroviral drug regimen against human immunodeficiency virus type-1 (HIV-1) replication. However, the low genetic barriers to drug-resistance or cross-resistance, side effects, as well as the unaffordable cost of NNRTIs compromise their clinical usage. Therefore, to develop novel NNRTIs with potent antiviral activity against HIV-1 becomes a major concern in the treatment and prevention of HIV/AIDS. (+)-Calanolide A, which is a natural product initially extracted from the tropical rainforest tree Calophyllum lanigerum, was identified as an attractive NNRTI against HIV-1 despite virus strains containing drug-resistant K103N/Y181C mutations. In this study, a chemical library was constructed based on the three chiral carbon centers of (+)-Calanolide A. After screening the activity against HIVNL4-3 wild-type and several NNRTI-resistant pseudoviruses, a small molecule 10-chloromethyl-11- demethyl-12-oxo-calanolide A (F18) was identified as novel NNRTI with promising anti-HIV efficacy. Further studies were performed to investigate the antiviral breadth, drug resistance profile and underlying mechanism of the action of F18. F18 consistently displayed a potent activity against primary HIV-1 isolates including various subtypes of M group, CRF01_AE, and laboratory-adapted drug-resistant viruses in PBMC based assay. Moreover, F18 displayed distinct profiles against 17 NNRTI-resistant pseudoviruses, with an excellent potency especially against one of the most prevalent strains with the Y181C mutation (EC50=1.0nM) in cell line based assay, which was in stark contrast from the extensively used NNRTIs nevirapine and efavirenz. F18-resistant viruses were induced by in vitro serial passages, and mutation L100I was appeared to be the dominant contributor to F18-resistance, further suggesting a binding motif different from nevirapine and efavirenz. The efficacy of F18 was non-antagonistic when used in combination with other antiretrovirals against both wild-type and drug-resistant viruses in infected PBMCs. Interestingly, F18 displayed a highly synergistic antiviral effect with nevirapine against nevirapine-resistant virus (Y181C). Furthermore, in silico docking analysis suggested that F18 may bind to the HIV-1 reverse transcriptase in a way different to other NNRTIs. For the potential as an anti-HIV-1 microbicide, F18 also showed the stable and rapid release, as well as the sustained antiviral activity against HIV-1 wild-type virus in a formulation temperature-sensitive acidic gel. In summary, this study presents F18 as a new potential drug for clinical use and also underlies new mechanism-based design for future NNRTI.
dc.description.nature	published_or_final_version
dc.description.thesisdiscipline	Microbiology
dc.description.thesislevel	doctoral
dc.description.thesisname	Doctor of Philosophy
dc.identifier.doi	http://dx.doi.org/10.5353/th_b4724650
dc.identifier.hkul	b4724650
dc.language	eng
dc.publisher	The University of Hong Kong (Pokfulam, Hong Kong)
dc.relation.ispartof	HKU Theses Online (HKUTO)
dc.rights	The author retains all proprietary rights, (such as patent rights) and the right to use in future works.
dc.rights	Creative Commons: Attribution 3.0 Hong Kong License
dc.source.uri	http://hub.hku.hk/bib/B47246509
dc.subject.lcsh	AIDS (Disease) - Chemotherapy.
dc.subject.lcsh	HIV infections - Chemotherapy.
dc.subject.lcsh	Reverse transcriptase - Inhibitors - Therapeutic use.
dc.subject.lcsh	Antiretroviral agents.
dc.title	Mechanism study of a small molecule F18 as a novel anti-HIV-1 non-nucleoside reverse transcriptase inhibitor
dc.type	PG_Thesis

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(+)-Calanolide A, which is a natural product initially extracted from the tropical rainforest tree Calophyllum lanigerum, was identified as an attractive NNRTI against HIV-1 despite virus strains containing drug-resistant K103N/Y181C mutations. In this study, a chemical library was constructed based on the three chiral carbon centers of (+)-Calanolide A. After screening the activity against HIVNL4-3 wild-type and several NNRTI-resistant pseudoviruses, a small molecule 10-chloromethyl-11- demethyl-12-oxo-calanolide A (F18) was identified as novel NNRTI with promising anti-HIV efficacy.

Further studies were performed to investigate the antiviral breadth, drug resistance profile and underlying mechanism of the action of F18. F18 consistently displayed a potent activity against primary HIV-1 isolates including various subtypes of M group, CRF01_AE, and laboratory-adapted drug-resistant viruses in PBMC based assay. Moreover, F18 displayed distinct profiles against 17 NNRTI-resistant pseudoviruses, with an excellent potency especially against one of the most prevalent strains with the Y181C mutation (EC50=1.0nM) in cell line based assay, which was in stark contrast from the extensively used NNRTIs nevirapine and efavirenz. F18-resistant viruses were induced by in vitro serial passages, and mutation L100I was appeared to be the dominant contributor to F18-resistance, further suggesting a binding motif different from nevirapine and efavirenz. The efficacy of F18 was non-antagonistic when used in combination with other antiretrovirals against both wild-type and drug-resistant viruses in infected PBMCs. Interestingly, F18 displayed a highly synergistic antiviral effect with nevirapine against nevirapine-resistant virus (Y181C). Furthermore, in silico docking analysis suggested that F18 may bind to the HIV-1 reverse transcriptase in a way different to other NNRTIs. For the potential as an anti-HIV-1 microbicide, F18 also showed the stable and rapid release, as well as the sustained antiviral activity against HIV-1 wild-type virus in a formulation temperature-sensitive acidic gel. 

In summary, this study presents F18 as a new potential drug for clinical use and also underlies new mechanism-based design for future NNRTI.</description.abstract><language>eng</language><publisher>The University of Hong Kong (Pokfulam, Hong Kong)</publisher><relation.ispartof>HKU Theses Online (HKUTO)</relation.ispartof><rights>The author retains all proprietary rights, (such as patent rights) and the right to use in future works.</rights><rights>Creative Commons: Attribution 3.0 Hong Kong License</rights><source.uri>http://hub.hku.hk/bib/B47246509</source.uri><subject.lcsh>AIDS (Disease) - Chemotherapy.</subject.lcsh><subject.lcsh>HIV infections - Chemotherapy.</subject.lcsh><subject.lcsh>Reverse transcriptase - Inhibitors - Therapeutic use.</subject.lcsh><subject.lcsh>Antiretroviral agents.</subject.lcsh><title>Mechanism study of a small molecule F18 as a novel anti-HIV-1 non-nucleoside reverse transcriptase inhibitor</title><type>PG_Thesis</type><identifier.hkul>b4724650</identifier.hkul><description.thesisname>Doctor of Philosophy</description.thesisname><description.thesislevel>doctoral</description.thesislevel><description.thesisdiscipline>Microbiology</description.thesisdiscipline><description.nature>published_or_final_version</description.nature><identifier.doi>10.5353/th_b4724650</identifier.doi><date.hkucongregation>2012</date.hkucongregation><bitstream.url>http://hub.hku.hk/bitstream/10722/146137/3/FullText.pdf</bitstream.url></item>