Binary pore-forming bacterial toxins are among Nature's most potent biological weapons with no antitoxins developed to combat them. Therefore, discovery of novel antitoxins is a high priority task in biomedical research. To secure a rapid counteracting response, these antitoxins are expected to be broad-spectrum, targeting a variety of toxic agents. In search for such universal compounds, we focus on those common intoxication steps that could be directionally targeted. Formation of the ion channels is the targetable universal property we explore. The objective of this proposal is to determine the mechanism of blockage of pore-forming binary bacterial toxins by polyvalent compounds, paying special attention to a fundamentally new group of blockers - cationic dendrimers. We hypothesize that due to the striking functional similarities in the transmembrane toxin uptake, the mechanism of their dendrimer-induced inhibition is universal. We build this hypothesis on our preliminary data, which showed that the cationic dendrimers effectively block pore-forming components of the toxins and protect Vero cell from toxin-triggered rounding. The rationale for this proposal is that once the mechanism of the polyvalent compounds docking into the channels is characterized, this will enable for further design of new classes of more effective antitoxins. We propose the following research plan:
Aim 1. Identify and interpret the physicochemical characteristics of the most effective cationic blockers (small-molecule, cyclodextrins and dendrimers).
Aim 2. Directly investigate the inhibitory effect of the cationic blockers on channel-facilitated uptake of enzymatic A components of the toxins.
Aim 3. Determine the molecular mechanisms of the toxin's inhibition. Under the 1st aim, planar lipid bilayer technique will be used to establish a structure-activity relationship and investigate the molecular details of blocker/pore binding reaction at the precise level of single-channel single-molecule interactions. Under the 2nd aim, using multi-channel bilayer measurements and cell assay, we will investigate if protective effect of the cationic blockers depends upon the decreased toxin uptake across the membrane. Under the 3rd aim, using molecular modeling and the theory of channel-facilitated membrane transport, we will describe a biophysical model of the blocker/pore interaction. The method for lead compound optimization we develop involves single-molecule characterization of polyvalent blockers of an opposite charge than the target pores and fine-tuning of the blocker/pore binding reaction parameters. The further development of this strategy represents an innovative approach that allows for the highly efficient identification of drugs using orders of magnitude fewer initial screening candidates compared to the standard """"""""trial and error"""""""" approach. The proposed research is significant, because it leads to a development of new drug discovery approaches that, in line with the NIAID """"""""Next generation research agenda,"""""""" will be based on design of broad-spectrum drugs versus traditional """"""""one bug - one drug"""""""" approach.
Upon the successful completion of the proposed project, fundamentally novel application of dendrimers as the broad-spectrum pore-blocking antitoxins for the treatment of anthrax and certain clostridium-associated diseases will be suggested and universal biophysical model of the blocker-induce channel inhibition will be developed. We expect that this study will have an important positive impact because it enables to advance a central step in our original approach to the rational design of the pore-blocking antitoxins - exploiting molecular features of the target molecule. On a broader scope, this proposal is in line with the NIAID Strategic Plan for Biodefense Research, searching for new paradigms and broad-spectrum strategies that will allow for more rapid and cost-effective development of countermeasures against emerging catastrophic biological threats, both natural and artificially engineered.
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