Inhibitory effect of novel amino acid and dipeptide-linked chitosan oligomers against HIV-1 infection
- Alternative Title
- 아미노산과 디펩타이드가 결합된 신규 키토올리고머의 HIV-1 감염억제 효과
- Abstract
- Chitosan derived substances including chitosan oligomers (COS) have been proposed for pharmaceutical applications due to their biocompatibility, non-toxicity and abundance in nature. As a result, several studies have been conducted regarding the bioactivity of chitosan oligomers and their derivatives. Hence, both chitosan oligomers and derived biomaterials are known to possess many biological activities such as antibacterial, antioxidant, anti-diabetic and anti-HIV. Similarly, numerous researches have been reported variety of bioactivities for natural peptides. Therefore, in order to form new series of compounds with enhanced bioactivity, chitosan oligomer conjugated amino acids and dipeptides were synthesized as a part of this study. Firstly, chitosan oligomers were linked to twenty naturally occurring amino acids and screened for their anti-HIV activity by syncytia formation and cell viability assays. Among all, COS-Aspartic acid (COS-D), COS-Glutamic acid (COS-E), COS-Asparagine (COS-N), COS-Threonine (COS-T) and COS-Tyrosine (COS-Y) showed strong protection against HIV-1 infection. Accordingly, COS were able to be linked to dipeptides formed by amino acids of active compounds from screening results. Hence, COS-Threonine-Aspartic acid (COS-TD) and COS-Threonine-Tyrosine (COS-TY) were obtained and screened for anti-HIV effect as well. Next, the mechanism underlying the anti-HIV effect of newly synthesized COS conjugates has been elucidated through several assays in vitro including syncytia formation, cell viability, co-cultivation, reverse transcriptase and protease inhibition, membrane-virus binding and Western blotting and PCR of HIV-related proteins using human T-cell lines. Among all, COS-D, COS-T were only able to show their anti-HIV effect through RT inhibition. COS-E was the only active compound to show HIV-1 protease inhibitory activity. COS-N and COS-Y were exhibited to effect the cell membrane-virus interaction rather than showing any enzyme inhibitory activity. Moreover, results clearly promoted COS-TY and COS-TD as the most active compounds. In comparison to others, COS-TD and COS-TY were able to protect cells from HIV infection by disrupting the virus-membrane interaction followed by RT inhibitory activity while amino acid linked COS were only effective against one stage of viral cycle. COS-TD and COS-TY surprisingly inhibited the expression of HIV-1 genomic products as well.
As a consequence, both chitosan oligomers and peptides are two important pharmacophores and when their synergism was combined towards bioactive properties, promising new agents could be developed for HIV treatment. In this study, it has been clearly showed that the dipeptide linked COS notably exhibited anti-HIV-1 properties. Further studies for enhancing the possibility of stopping HIV-1 in every stage of viral cycle will promote new insights into proper control and treatment HIV-1 infection
- Issued Date
- 2012
- Awarded Date
- 2012. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Table of Contents
Abstract i
Table of Contents iii
List of Figures vii
List of Tables x
List of Abbreviation xi
Chapter I. General Introduction 1
Chapter II. Synthesis and Screening of Amino acid-conjugated Chitosan Oligomers for Anti-HIV activity
1. Introduction 8
1.1. Chitosan oligomers and their bioavailability 8
1.2. Chitosan oligomer derivatization 9
1.3. Conjugation of amino acids and peptides 10
2. Materials and Methods 12
2.1. Materials and Chemicals for Synthesis 12
2.2. Materials and Chemicals for Anti-HIV Screening 12
2.3. Synthesis of amino acid and dipeptide conjugated chitosan oligomers 12
2.4. Syncytia formation assay 16
2.5. Cell viability assay 19
3. Results and Discussion 20
3.1. Synthesis of amino-acid conjugated chitosan oligomers 22
3.2. Screening activity against HIV-1 infection 67
3.2.1. Protection of cells from lytic effects of HIV-1 68
3.2.2. Screening activity for syncytia formation inhibition 70
3.3. Anti-HIV-1 screening of dipeptide conjugated chitosan oligomers 73
Chapter III. Elucidation of the action mechanism against HIV-1 infection
1. Introduction 79
1.1. HIV-1. 79
1.1.1. The structure of HIV-1 79
1.1.2. The viral envelope 79
1.1.3. The virion 82
1.1.4. Classification of HIV 82
1.1.5. Current status of HIV/AIDS in Korea 86
1.1.6. HIV replication 86
1.1.7. Course of HIV infection 94
1.2. The four stages of HIV disease 95
1.2.1. Acute infection stage 95
1.2.2. Asymptomatic stage 96
1.2.3. Early HIV disease 96
1.2.4. Advanced HIV disease 97
1.3. Polysaccharides as anti-HIV Agents 98
1.4. Chitosan and chitosan oligomers (COS) as bioactive materials 99
1.5. Peptides as anti-HIV agents 100
2. Materials and Methods 102
2.1. Materials and chemicals 102
2.2. Cell lines and virus 102
2.3. Cell culture 103
2.4. Determination of lytic effect of HIV-1 104
2.5. Determination of syncytia formation 104
2.6. Co-culture assay 104
2.7. Delayed addition of compounds to HIV-1IIIB infected CEM-SS cells 105
2.8. p24 ELISA assay 105
2.9. Western blot analysis 106
2.10. In vitro reverse transcriptase activity assay 107
2.11. RNA extraction 107
2.12. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) 108
2.13. HIV-1 protease inhibition assay 112
2.14. Luciferase and β-galactosidase gene reporter assay 112
2.15. Statistical analysis 113
3. Results and Discussion 113
3.1. Determination of proposed action mechanism of samples 116
3.1.1. Syncytium Count 116
3.1.2. Reverse Transcriptase Inhibition 118
3.1.3. Protease Inhibition 122
3.1.4. Proposed mechanism of action of the compounds 125
3.2. COS-D 127
3.3. COS-Y 130
3.4. COS-N 132
3.5. COS-T 134
3.6. COS-E 136
3.7. COS-TD and COS-TY 139
3.7.1. Inhibition of HIV-1 infection 143
3.7.2. Inhibition of HIV-1 viral particle production 147
3.7.3. Co-cultivation assays 150
3.7.4. Delayed addition of COS-TD and COS-TY 154
3.7.5. Gene Reporter Assay 154
Summary 158
References 161
- Degree
- Doctor
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