Crystallographic studies of the complexes of antiviral protein griffithsin with glucose and N-acetylglucosamine.
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ABSTRACT:
SUBMITTER: Ziolkowska NE
PROVIDER: S-EPMC2206701 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mode of binding of oligosaccharides to griffithsin, an antiviral lectin from the red alga Griffithsia sp., was investigated by a combination of X-ray crystallography, isothermal titration calorimetry, and molecular modeling. The structures of complexes of griffithsin with 1-->6alpha-mannobiose and with maltose were solved and refined at the resolution of 2.0 and 1.5 A, respectively. The thermodynamic parameters of binding of 1-->6alpha-mannobiose, maltose, and mannose to griffithsin were determined. Binding profiles of 1-->6alpha-mannobiose and mannose were similar with Kd values of 83.3 microM and 102 microM, respectively. The binding of maltose to griffithsin was significantly weaker, with a fourfold lower affinity (Kd = 394 microM). In all cases the binding at 30 degrees C was entropically rather than enthalpically driven. On the basis of the experimental crystal structures, as well as on previously determined structures of complexes with monosaccharides, it was possible to create a model of a tridentate complex of griffithsin with Man9GlcNAc2, a high mannose oligosaccharide commonly found on the surface of viral glycoproteins. All shorter oligomannoses could be modeled only as bidentate or monodentate complexes with griffithsin. The ability to mediate tight multivalent and multisite interactions with high-mannose oligosaccharides helps to explain the potent antiviral activity of griffithsin.
| S-EPMC7167611 | biostudies-literature
Project description:Application of plant-based protein expression systems for bulk production of recombinant protein pharmaceuticals is building momentum. There are considerable regulatory challenges to consider in commercialization of plant-made pharmaceuticals (PMPs), some of which are inherent to plant-production systems and others that are common with other production systems, but are new to PMPs because of the youth of the industry. In this review, we discuss our recent and ongoing experience with bulk production of the HIV microbicide candidate, griffithsin (GRFT), utilizing plant-based transient protein expression, with specific focus on areas relevant to commercial manufacturing of bulk GRFT active pharmaceutical ingredient (API). Analytical programs have been developed for the qualification and monitoring of both the expression vector system and the API detailing our experience and plans for each. Monitoring postpurification protein modifications are discussed in relation to stability and safety programs. Expression, processing and analytics programs are associated with increased manufacturing costs in current good manufacturing practice (cGMP) production because of the required qualification testing. The impact of these costs on the overall cost of goods is particularly relevant to GRFT manufacturing because GRFT, as an HIV microbicide, is most needed in populations at high risk for HIV exposure in resource-poor countries. Consequently, GRFT for microbicide applications is a very cost-sensitive recombinant PMP. We have therefore emphasized maintaining a low cost of goods. We provide a review of the literature on the economics of PMPs with various expression systems and how they may impact production costs and complexity.
| S-EPMC5016770 | biostudies-literature
Project description:Griffithsin (GRFT), an algae-derived lectin, is one of the most potent viral entry inhibitors discovered to date. It is currently being developed as a microbicide with broad-spectrum activity against several enveloped viruses. GRFT can inhibit human immunodeficiency virus (HIV) infection at picomolar concentrations, surpassing the ability of most anti-HIV agents. The potential to inhibit other viruses as well as parasites has also been demonstrated. Griffithsin's antiviral activity stems from its ability to bind terminal mannoses present in high-mannose oligosaccharides and crosslink these glycans on the surface of the viral envelope glycoproteins. Here, we review structural and biochemical studies that established mode of action and facilitated construction of GRFT analogs, mechanisms that may lead to resistance, and in vitro and pre-clinical results that support the therapeutic potential of this lectin.
| S-EPMC5086628 | biostudies-literature
Project description:Hepatitis C virus (HCV)-infected patients undergoing liver transplantation universally experience rapid reinfection of their new liver graft. Current treatment protocols do not prevent graft reinfection and, in addition, an accelerated disease progression is observed. In the present study, we have evaluated a novel strategy to prevent HCV infection using a lectin, griffithsin (GRFT) that specifically binds N-linked high-mannose oligosaccharides that are present on the viral envelope. The antiviral effect of GRFT was evaluated in vitro using the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems. We show here that preincubation of HCVpp and HCVcc with GRFT prevents infection of Huh-7 hepatoma cells. Furthermore, GRFT interferes with direct cell-to-cell transmission of HCV. GRFT acts at an early phase of the viral life cycle by interfering in a genotype-independent fashion with the interaction between the viral envelope proteins and the viral receptor CD81. The capacity of GRFT to prevent infection in vivo was evaluated using uPA(+/+)-SCID mice (uPA stands for urokinase-type plasminogen activator) that harbor human primary hepatocytes in their liver (chimeric mice). In this proof-of-concept trial, we demonstrated that GRFT can mitigate HCV infection of chimeric mice. Treated animals that did become infected demonstrated a considerable delay in the kinetics of the viral infection. Our data demonstrate that GRFT can prevent HCV infection in vitro and mitigate HCV infection in vivo. GRFT treatment of chronically infected HCV patients undergoing liver transplantation may be a suitable strategy to prevent infection of the liver allograft.
| S-EPMC3194994 | biostudies-literature
Project description:It is essential that mRNA-binding proteins recognize specific motifs in target mRNAs to control their processing, localization, and expression. Although mRNAs are typically targets of many different regulatory factors, our understanding of how they work together is limited. In some cases, RNA-binding proteins work cooperatively to regulate an mRNA target. A classic example is Drosophila melanogaster Pumilio (Pum) and Nanos (Nos). Pum is a sequence-specific RNA-binding protein. Nos also binds RNA, but interaction with some targets requires Pum to bind first. We recently determined crystal structures of complexes of Pum and Nos with two different target RNA sequences. A crystal structure in complex with the hunchback mRNA element showed how Pum and Nos together can recognize an extended RNA sequence with Nos binding to an A/U-rich sequence 5' of the Pum sequence element. Nos also enables recognition of elements that contain an A/U-rich 5' sequence, but imperfectly match the Pum sequence element. We determined a crystal structure of Pum and Nos in complex with the Cyclin B mRNA element, which demonstrated how Nos clamps the Pum-RNA complex and enables recognition of the imperfect element. Here, we describe methods for expression and purification of stable Pum-Nos-RNA complexes for crystallization, details of the crystallization and structure determination, and guidance on how to analyze protein-RNA structures and evaluate structure-driven hypotheses. We aim to provide tips and guidance that can be applied to other protein-RNA complexes. With hundreds of mRNA-binding proteins identified, combinatorial control is likely to be common, and much work remains to understand them structurally.
| S-EPMC6697268 | biostudies-literature
Project description:The crystal structure of griffithsin, an antiviral lectin from the red alga Griffithsia sp., was solved and refined at 1.3 A resolution for the free protein and 0.94 A for a complex with mannose. Griffithsin molecules form a domain-swapped dimer, in which two beta strands of one molecule complete a beta prism consisting of three four-stranded sheets, with an approximate 3-fold axis, of another molecule. The structure of each monomer bears close resemblance to jacalin-related lectins, but its dimeric structure is unique. The structures of complexes of griffithsin with mannose and N-acetylglucosamine defined the locations of three almost identical carbohydrate binding sites on each monomer. We have also shown that griffithsin is a potent inhibitor of the coronavirus responsible for severe acute respiratory syndrome (SARS). Antiviral potency of griffithsin is likely due to the presence of multiple, similar sugar binding sites that provide redundant attachment points for complex carbohydrate molecules present on viral envelopes.
| S-EPMC7126681 | biostudies-literature
Project description:This review focuses on the important contributions that macromolecular crystallography has made over the past 12 years to elucidating structures and mechanisms of the essential proteases of coronaviruses, the main protease (M(pro) ) and the papain-like protease (PL(pro) ). The role of X-ray crystallography in structure-assisted drug discovery against these targets is discussed. Aspects dealt with in this review include the emergence of the SARS coronavirus in 2002-2003 and of the MERS coronavirus 10 years later and the origins of these viruses. The crystal structure of the free SARS coronavirus M(pro) and its dependence on pH is discussed, as are efforts to design inhibitors on the basis of these structures. The mechanism of maturation of the enzyme from the viral polyprotein is still a matter of debate. The crystal structure of the SARS coronavirus PL(pro) and its complex with ubiquitin is also discussed, as is its orthologue from MERS coronavirus. Efforts at predictive structure-based inhibitor development for bat coronavirus M(pro) s to increase the preparedness against zoonotic transmission to man are described as well. The paper closes with a brief discussion of structure-based discovery of antivirals in an academic setting.
| S-EPMC7163996 | biostudies-literature
Project description:Post-translational modifications, including O-GlcNAcylation, play fundamental roles in modulating cellular events, including transcription, signal transduction, and immune signaling. Several molecular targets of O-GlcNAcylation associated with pathogen-induced innate immune responses have been identified; however, the direct regulatory mechanisms linking O-GlcNAcylation with antiviral RIG-I-like receptor signaling are not fully understood. In this study, we found that cellular levels of O-GlcNAcylation decline in response to infection with Sendai virus. We identified a heavily O-GlcNAcylated serine-rich region between amino acids 249-257 of the mitochondrial antiviral signaling protein (MAVS); modification at this site disrupts MAVS aggregation and prevents MAVS-mediated activation and signaling. O-GlcNAcylation of the serine-rich region of MAVS also suppresses its interaction with TRAF3; this prevents IRF3 activation and production of interferon-β. Taken together, these results suggest that O-GlcNAcylation of MAVS may be a master regulatory event that promotes host defense against RNA viruses.
| S-EPMC7884448 | biostudies-literature
Project description:Griffithsin is a lectin with potent antiviral activity against enveloped viruses. The objective of this study was to assess Griffithsin's inhibitory effect on porcine epidemic diarrhea virus (PEDV). The results showed that Griffithsin reduced PEDV infection of Vero cells by approximately 82.8%. Moreover, using time-of-addition assays and RT-qPCR, we found that delayed addition of Griffithsin had a weaker inhibitory effect on PEDV than earlier treatment. The mechanism of Griffithsin's action against PEDV involved both preventing viral attachment to host cells and disrupting cell-to-cell transmission; its dual mode of action distinguished Griffithsin from most other antiviral drugs. In conclusion, Griffithsin was identified as a potent PEDV inhibitor and may represent a candidate drug for preventing PEDV infection.
| S-EPMC7089098 | biostudies-literature
Project description:There is a rising global incidence of Candida strains with high levels of resistance to fluconazole and other antifungal drugs, hence the need for novel antifungal treatment strategies. Here, we describe the first evidence of antifungal activity of Q-Griffithsin (Q-GRFT), a recombinant oxidation-resistant variant of Griffithsin, a marine red algal lectin with broad-spectrum antiviral activity. We demonstrated that Q-GRFT binds to α-mannan in the Candida albicans cell wall. We also observed that Q-GRFT binding disrupted cell wall integrity and induced reactive oxidative species (ROS) formation, resulting in cell death. Furthermore, we showed that Q-GRFT inhibited the growth of other Candida species C. glabrata, C. parapsilosis, and C. krusei and had modest activity against some strains of multi- and pandrug-resistant C. auris. We found that Q-GRFT induced differential expression of numerous genes involved in response to cell stress, including those responsible for neutralizing ROS production and cell cycle regulation. In conclusion, this novel antifungal activity suggests that Q-GRFT is potentially an ideal drug candidate and represents an alternative strategy for the prevention and treatment of candidiasis. IMPORTANCE Fungal infections contribute to morbidity and mortality annually, and the number of organisms that are nonresponsive to the current available drug regimens are on the rise. There is a need to develop new agents to counter these infections and to add to the limited arsenal available to treat fungal infections. Our study has identified Q-GRFT, a broad-spectrum antiviral protein that harbors growth-inhibitory activity against several Candida strains, as a potential candidate for the prevention and treatment of fungal infections.
| S-EPMC8557872 | biostudies-literature