TY - JOUR
T1 - Exploring the chemical space around 8-mercaptoguanine as a route to new inhibitors of the folate biosynthesis enzyme HPPK
AU - Chhabra, Sandeep
AU - Barlow, Nicholas
AU - Dolezal, Olan
AU - Hattarki, Meghan
AU - Newman, Janet
AU - Peat, Thomas
AU - Graham, Bimbil
AU - Swarbrick, James David
PY - 2013
Y1 - 2013
N2 - As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (K-D similar to 13 mu M), inhibited the S. aureus enzyme (SaHPPK) (IC50 similar to 41 mu M), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N-9 substitution, or tautomerization arising from N-7 substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N-9-H center dot center dot center dot Val46 hydrogen bond, combined with the limited space available around the N-9 position. The water-filled pocket under the N-7 position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N-7 (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (K-D similar to 12 mu M). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 angstrom X-ray structure of the SaHPPK/21a complex confirms that extension from the N-7 position towards the Mg2+-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg2+ centres or Mg2+-binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.
AB - As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg2+-dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (K-D similar to 13 mu M), inhibited the S. aureus enzyme (SaHPPK) (IC50 similar to 41 mu M), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N-9 substitution, or tautomerization arising from N-7 substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N-9-H center dot center dot center dot Val46 hydrogen bond, combined with the limited space available around the N-9 position. The water-filled pocket under the N-7 position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N-7 (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (K-D similar to 12 mu M). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 angstrom X-ray structure of the SaHPPK/21a complex confirms that extension from the N-7 position towards the Mg2+-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg2+ centres or Mg2+-binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.
UR - http://search.proquest.com.ezproxy.lib.monash.edu.au/docview/1330900778?accountid=12528
U2 - 10.1371/journal.pone.0059535
DO - 10.1371/journal.pone.0059535
M3 - Article
VL - 8
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 4
M1 - e59535
ER -