Exploring novel strategies for AIDS protozoal pathogens: α-helix mimetics targeting a key allosteric protein–protein interaction in C. hominis thymidylate synthase-dihydrofolate reductase (TS-DHFR)

Abstract

The bifunctional enzyme thymidylate synthase–dihydrofolate reductase (TS–DHFR) from the protozoal parasite Cryptosporidium hominis is a potential molecular target for the design of antiparasitic therapies for AIDS-related opportunistic infections. The enzyme exists as a homodimer with each monomer containing a unique swap domain known as a “crossover helix” that binds in a cleft on the adjacent DHFR active site. This crossover helix is absent in species containing monofunctional forms of DHFR such as human. An in-depth understanding of protein–protein interactions between the crossover helix and adjacent DHFR active site that might modulate enzyme integrity or function would allow for insights into rational design of species-specific allosteric inhibitors. Mutational analysis coupled with structural studies and biophysical and kinetic characterization of crossover helix mutants identifies this domain as essential for full enzyme stability and catalytic activity, and pinpoints these effects to distinct faces of the crossover helix important in protein–protein interactions. Moreover, targeting this helical protein interaction with α-helix mimetics of the crossover helix leads to selective inhibition and destabilization of the C. hominis TS–DHFR enzyme, thus validating this region as a new avenue to explore for species-specific inhibitor design.

Bioinformatics Applications on Cryptosporidium Research: A Review

“…With respect to the development of potential anti-Cryptosporidium target to this enzyme, both non-conserved TS and DHFR residues can be mutated that eventually give negative regulation prior to multiple steps in its catalytic cycle 41 . Thus, TS-DHFR enzyme belongs to C. hominis could be a potential target for the design of anti-Cryptosporidium agent for those affected people with weak immunity like AIDS-related immunocompromised patients 82 . Interestingly, TS-DHFR enzyme from C. hominis has unique feature of a homodimer which its monomer has a crossover helix that interact with adjacent cleft on the other active site within this enzyme 41,82 .…”

Understanding the molecular mechanism of substrate channeling and domain communication in protozoal bifunctional TS-DHFR

“…6A) (Kumar et al, 2013(Kumar et al, , 2014Mukerjee et al, 2015). The crossover helical region of the Ch TS-DHFR, shown to be important for DHFR catalysis, was successfully targeted with ɑ-helical mimetics (Vargo et al, 2009;Martucci et al, 2013) (Fig. 6B).…”

Helix mimetics: Recent developments

“…Although it is natural to develop new methodologies on tried and trusted model systems, this should change and several recent studies provide encouragement. Inhibitors of HIF-1a/p300 (Lao et al, 2014a(Lao et al, , 2014bBurslem et al, 2014), ER/co-activator, (Becerril and Hamilton, 2007;Ravindranathan et al, 2013) (Martucci et al, 2013) and amyloid aggregation (hIAPP) (Hebda et al, 2009) have been described. The hIAPP aggregation inhibitors have an unusual mode of action in that they are proposed to sequester a helical intermediate en route to the bstrand conformer that assembles into b-sheet fibrils.…”