Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design

Karginov, Vladimir A.; Nestorovich, Ekaterina M.; Moayeri, Mahtab; Leppla, Stephen H.; Bezrukov, Sergey M.

doi:10.1073/pnas.0507488102

Cited by 111 publications

(166 citation statements)

References 31 publications

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“…2A). Fischer 344 rats (n ϭ 5) were challenged with LeTx to evaluate the in vivo neutralization potential of the hMAbs, as previously described (29). Consistent with the in vitro results, 22F1 showed the highest level of protection and resulted in 100% survival even when mixed with PA at the molar ratio of 1:3 (data not shown).…”

Section: Resultssupporting

confidence: 60%

Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine

Chi

Liu

et al. 2015

Clin. Vaccine Immunol.

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The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design.

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Section: Resultssupporting

confidence: 60%

Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine

Chi

Liu

et al. 2015

Clin. Vaccine Immunol.

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“…EF is an adenylate cyclase, and LF is a protease that cleaves mitogen-activated protein kinase kinases. The enzymatic activities of these proteins contribute to disease progression in several ways and at different stages of infection (16).Several anthrax-toxin inhibitors have been described that interfere with different steps in this intoxication pathway (8,11,12,16,(20)(21)(22)(23)(24)(25)(26), but none has targeted the host receptors. Here, we describe the development of a polyvalent receptor-directed anthrax-toxin inhibitor that binds both receptors and protects animals from toxin challenge.…”

mentioning

confidence: 99%

Polyvalent inhibitors of anthrax toxin that target host receptors

Basha

Rai

Poon

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Resistance of pathogens to antimicrobial therapeutics has become a widespread problem. Resistance can emerge naturally, but it can also be engineered intentionally, which is an important consideration in designing therapeutics for bioterrorism agents. Blocking host receptors used by pathogens represents a powerful strategy to overcome this problem, because extensive alterations to the pathogen may be required to enable it to switch to a new receptor that can still support pathogenesis. Here, we demonstrate a facile method for producing potent receptor-directed antitoxins. We used phage display to identify a peptide that binds both anthraxtoxin receptors and attached this peptide to a synthetic scaffold. Polyvalency increased the potency of these peptides by >50,000-fold in vitro and enabled the neutralization of anthrax toxin in vivo. This work demonstrates a receptor-directed anthrax-toxin inhibitor and represents a promising strategy to combat a variety of viral and bacterial diseases.antimicrobial resistance ͉ phage display ͉ therapeutics P athogens can develop resistance to drugs directed against microbial targets by modifying the drug, by lowering the concentration of drug that reaches the target, or by mutating the target (1, 2). There is also an increasing concern that therapeutics developed for bioterrorism agents may be rendered ineffective if the microbial target is altered intentionally. This problem could be overcome, however, by designing inhibitors that block host proteins used by the pathogen or its toxins to cause disease.Microbial pathogens and their products interact with host structures to facilitate colonization or to promote cellular uptake. Many of these interactions are polyvalent, meaning that they involve the simultaneous binding of multiple ligands on one entity to multiple receptors on another (3). The design of synthetic polyvalent (4-8) or oligovalent (9, 10) molecules also represents a promising approach to enhance the potency of inhibitors of microbial pathogens and toxins. Current examples of this approach have involved the design of molecules that bind directly to the pathogen or toxin. Inhibitors that bind host proteins would represent an effective way to attenuate virulence that may be less susceptible to resistance mechanisms, and the use of polyvalency could provide a significant enhancement in the potency of these inhibitors.ANTXR1 and ANTXR2 are host receptors that bind and internalize anthrax toxin (11,12). These proteins are likely important for anthrax pathogenesis because the toxin impairs the immune response and is responsible for the major symptoms and death associated with anthrax. Thus, blocking these receptors could represent a promising approach to anthrax therapy.ANTXR1 and ANTXR2 are widely expressed type I membrane proteins that bind components of the extracellular matrix (13). They both contain an extracellular I domain, which binds the protective antigen (PA) component of anthrax toxin. The two proteins are 40% identical overall and share 60% identity within ...

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“…3 with the linear van't Hoff equation widely used for the thermodynamic analysis of binding reactions (4,10,11), ln K eq = ΔH=RT − ΔS=R; [1] where T and R are the absolute temperature and universal gas constant, respectively, and, if both enthalpy ΔH and entropy ΔS are assumed to be temperature-independent, we obtain ΔH ' 1 kcal/mol or about 2 k B T per molecule, where k B is the Boltzmann constant. The enthalpy term is supposed to characterize the strength of the blocker-channel interaction and be equal to the depth ΔU of the temperature-independent flat potential well for the blocking particle inside the channel (see model considerations below).…”

Section: Discussionmentioning

confidence: 99%

Kinetics and thermodynamics of binding reactions as exemplified by anthrax toxin channel blockage with a cationic cyclodextrin derivative

Nestorovich

Karginov

Berezhkovskii

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The thermodynamics of binding reactions is usually studied in the framework of the linear van't Hoff analysis of the temperature dependence of the equilibrium constant. The logarithm of the equilibrium constant is plotted versus inverse temperature to discriminate between two terms: an enthalpic contribution that is linear in the inverse temperature, and a temperature-independent entropic contribution. When we apply this approach to a particular case-blockage of the anthrax PA 63 channel by a multicharged cyclodextrin derivative-we obtain a nearly linear behavior with a slope that is characterized by enthalpy of about 1 kcal/mol. In contrast, from blocker partitioning between the channel and the bulk, we estimate the depth of the potential well for the blocker in the channel to be at least 8 kcal/mol. To understand this apparent discrepancy, we use a simple model of particle interaction with the channel and show that this significant difference between the two estimates is due to the temperature dependence of the physical forces between the blocker and the channel. In particular, we demonstrate that if the major component of blocker-channel interaction is van der Waals interactions and/or Coulomb forces in water, the van't Hoff enthalpy of the binding reaction may be close to zero or even negative, including cases of relatively strong binding. The results are quite general and, therefore, of importance for studies of enzymatic reactions, rational drug design, small-molecule binding to proteins, protein-protein interactions, and protein folding, among others.protein-ligand binding | molecular docking M otivated by the search for efficient small-molecule blockers of "virulent" transmembrane channels (1-3), we investigated the temperature behavior of the blockage reaction. The temperature dependence of the equilibrium constant of blocker association with the pore, that is, the reaction blocker(bulk) + channel(empty) ⇄ channel − blocker, was found to be surprisingly weak, with the temperature coefficient jQ 10 j ' 1.08. Applying the standard linear van't Hoff analysis to evaluate the enthalpy of an association reaction (4) to that of channel blockage (5), we arrive at an estimated enthalpy for the channel-blocker interaction of about 1 kcal/mol. However, examination of blocker partitioning between the bulk and the channel interior gives an estimate for the depth of the potential well for the blocker in the pore of at least 8 kcal/mol. Although these two estimates generally do not have to coincide, this impressive difference compelled us to take a detailed look at the assumptions that are often used in the thermodynamic analysis of blocking reactions as well as at the possible physical forces involved in the channel-blocker interaction. Our analysis is based on consideration of a simple model of particle interaction with the channel (6-8), which allows an explicit calculation of the involved thermodynamics. As might be expected, in the case of the temperatureindependent flat potential well, the enthalpy of the bindin...

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Blocking anthrax lethal toxin at the protective antigen channel by using structure-inspired drug design

Cited by 111 publications

References 31 publications

Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine

Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine

Polyvalent inhibitors of anthrax toxin that target host receptors

Kinetics and thermodynamics of binding reactions as exemplified by anthrax toxin channel blockage with a cationic cyclodextrin derivative

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