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- Johansson, Emil, 1985-
(författare)
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Tailored conjugates of N-acetylneuraminic acid and small molecules that block virus cell attachment and entry
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Viruses are obligate intracellular parasites, unable to replicate without exploiting machinery andmaterials from host cells. Pandemics of viral diseases have had large impacts on human socieities and are continued threats to global health. The most efficient means of controlling viral diseases are preventive measures such as immunization of the population, social distancing, and basic hygiene routines. Another mean is development of antiviral drugs that could be used as preventive measures and in treatment of infected individuals. Coxsackievirus A24 variant (CVA24v) is a highly contagious pathogen that cause large outbreaks and pandemics of the eye infection acute hemorrhagic conjunctivitis. Human adenovirus D species type 37 (HAdV-D37) causes epidemics of the severe eye infection epidemic keratoconjunctivitis, that can become life-threatening in immunocompromised individuals. Currently, no specific treatments (vaccine or antivirals) are available to combat the diseases caused by these two pathogens.CVA24v and HAdV-D37 bind to N-acetylneuraminic acid (Neu5Ac) glycans on host cells facilitating attachment and subsequent infection. In this thesis, we explored inhibition of this common recognition motif by development of pentavalent Neu5Ac containing molecules with radial topology to act as decoy receptors. This allowed us to study the potential of development of a general inhibitor targeting both these viruses. The developed compounds inhibited attachment of CVA24v and HAdVD37 to cells.Furthermore, we developed divalent Neu5Ac tools to validate if targeting the Neu5Acmediated attachment of CVA24v to cells were a potential target for antiviral drug discovery and development. The results from these studies indicate that development of a Neu5Ac-based antiviral targeting CVA24v looks bleak as the primary receptor utilized by this virus is ICAM-1. The work with developing Neu5Ac tools led to a side project with synthesis of 4-O-alkyl Neu5Ac analogs. In this project we provided a method to synthesize 4-O-alkyl analogs of Neu5Ac and gave insights into the scope of the reaction. This work could have have value in drug discovery.Targeting enterovirus uncoating is a well explored strategy for the inhibition of enterovirus infection. In this thesis, we synthezied novel branched probes of pleconaril (a well-known pocket binding molecule) to study if targeting the unique branched pocket of CVA24v could have potential as a target for antiviral drug discovery. Further experiments are needed to draw conclusions in regards to the future prospects of targeting this unique feature.At last, two novel classes of trivalent Neu5Ac conjugates were develop using a structure-based approach targeting HAdV-D37, -D36, and -D26. This led to a more potent compound towards HAdVD37 further validating that targeting the attachment of this virus to cells is a reasonable strategy for antiviral drug development. Towards HAdV-D26 the inhibitory effect was saturated at 50%, likely due to engagement of other receptors. Evaluation towards HAdV-D36 is currently ongoing. Structural biology studies, indicates the compounds bind to the viruses via chelation of their trimeric binding sites. Taken together, these compounds have potential to be used as chemical tools to study the biology of human adenoviruses and perhaps other Neu5Ac binding proteins.
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| 2. |
- Chandra, Naresh, 1987-
(författare)
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The glycobiology of human adenovirus infections : implications for tropism and treatment
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human adenoviruses (HAdVs) are common human pathogens, causing gastrointestinal, ocular, and respiratory infections on a regular basis. Epidemic keratoconjunctivitis (EKC) is a severe ocular infection for which no approved antivirals are available. HAdV-D37 is one of the causative agents of EKC and uses sialic acid (SA)-containing glycans as cellular receptors. HAdV-D37 interacts with SA via the knob domain of the trimeric virus fiber protein, containing three SA-binding sites. HAdV-D37 also bind to glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, using biochemical and cell-based assays, the impact of GAGs on HAdV-D37 infection (paper I) was investigated. We found that HAdV-D37 interacts with both soluble and cell-surface sulfated GAGs via the knob domain of the viral fiber protein. Remarkably, removal of heparan sulfate (HS; a type of GAG) from human corneal epithelial (HCE) cells by heparinase III enhanced HAdV-D37 infection. We propose that sulfated GAGs in bodily secretions and on plasma membranes function as decoy receptors that prevent the virus from binding to SA-containing receptors and inhibit subsequent virus infection. We also found abundant HS in the basement membrane of the human corneal epithelium. We suggest that this layer of HS functions as a barrier to sub-epithelial infection of HAdV-D37. Based on this finding, we hypothesized that GAG-mimetics may act as artificial decoy receptors and inhibit HAdV-D37 infection. Here, the antiviral effect of suramin (a known GAG-mimetic) and its analogs against HAdV-D37 (paper II) was evaluated. Interestingly, all compounds displayed antiviral effects by inhibiting the binding of HAdV-D37 to HCE cells. The antiviral effect of suramin was HAdV species-specific. We report for the first time that virus binding to cell-surface decoy receptor constitutes a potential target for antiviral drug development.HAdVs are the major cause of infectious conjunctivitis, constituting up to 75% of all conjunctivitis cases worldwide. Species B HAdV type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause EKC. Recently, HAdV-D53, -D54, and -D56 have emerged as new EKC-causing agents. HAdV-E4 causes both PCF and EKC. SA-containing glycans have been established as cellular receptors for HAdV-D37. By means of cell-based assays, we investigated if ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells (paper III). It was found that SA-containing glycans function as cellular receptors for five (HAdV-D8, -D37, -D53, -D54, and -D64) out of six EKC-causing species D HAdVs. We showed that these viruses interact with SAs via the knob domain of the viral fiber protein. HAdV-E4 and -D56 infection of cells was independent of SAs. Surprisingly, HCE cells were completely refractory to HAdV-B3 infection. A trivalent sialic acid (TSA) derivative ME0462 (compound 17a in paper II), designed to bind to SA-binding sites on HAdV-D37 fiber knob, also showed potent antiviral activity against several EKC-causing HAdVs. This suggests that ME0462 can be used as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs. Surface plasmon resonance (SPR) analysis confirmed a direct interaction between ME0462 and fiber knobs of EKC-causing HAdVs.Recently, a TSA derivative (ME0322; designed to bind to SA-binding sites on HAdV-D37 fiber knob) was shown potent antiviral against HAdV-D37 in vitro. To improve the antiviral potency of this compound, six new TSA derivatives were synthesized and their inhibitory effects were evaluated against HAdV-D37 (paper IV). Interestingly, the best compound 17a was found approximately three orders of magnitude more potent (IC50 (binding) = 1.4 nM, IC50 (infection) = 2.9 nM) than ME0322 (IC50 in µM range). SPR data showed that HAdV-D37 fiber knob binds to TSA compounds with high affinities. Structural data revealed the trivalent binding mode of all newly synthesized TSA compounds to HAdV-D37 fiber knob. Ophthalmic toxicity of compound 17a (best compound) was also investigated in rabbits without any sign of toxicity.HAdV-D36 is a member of species D HAdV and has the ability to infect a broad range of animals, which is unusual for HAdVs. Another remarkable feature of HAdV-D36 is that this virus induces obesity in experimental animals. Several epidemiological studies highlighted a link between HAdV-D36 and human obesity. There is no information about the cellular receptor usage by HAdV-D36. Using structural biology and cell-based approaches, we investigated the cellular receptor(s) for HAdV-D36 (paper V). We show that HAdV-D36 attaches to host cells (via the fiber knob) using the coxsackie and adenovirus receptor (CAR), SA-containing glycans, and one or more unknown proteins or glycoproteins. Using glycan microarray, we found that HAdV-D36 displays binding preference to a rare SA-variant: 4-O,5-N-diacetylneuraminic acid (Neu4,5Ac2), over the more common SA (in humans) i.e. 5-N-acetylneuraminic acid (Neu5Ac). Structural analysis of HAdV-D36 fiber knob:Neu4,5Ac2 complex explained this preference. To date, Neu4,5Ac2 has not been detected in humans, although it is synthesized by many domestic and livestock animals. Our results indicate that HAdV-D36 has evolved to utilize a specialized set of cellular receptors that coincide with a unique host range and pathogenicity profile.These studies provide insights into multiple roles of glycans in HAdV infection cycle and highlight the therapeutic potential of glycans/glycan-mimetics in HAdV-D37 infection.
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