Search

Galactofuranose (Galf) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5′-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galfin cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM–small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.

S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch.

The first iron complexes of the tetracationic 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetra-N-pyridiniumylporphyrin, (OEPy4P)4+, in which four pyridine molecules are attached to the meso-carbon atoms through their nitrogen atoms, were synthesized in three steps starting from Zn(OEP) (Zn-β-octaethylporphyrin) with an overall yield of ca. 30 %. The X-ray structure of [FeII(OEPy4P)(pyridine)2](CF3SO3)3(Br) established that the molecule adopts a severely distorted nonplanar saddle conformation. 1H NMR spectroscopy showed that the [FeII(OEPy4P)(B)2]4+ ligated complexes, B = pyridine (Py) or imidazole (Im), are low-spin, FeII, S = 0. These complexes exhibit redox potentials for the FeIII/FeII couple that range between 0.62 and 0.38 V (vs. Ag/AgCl) and are shifted by as much as +0.7 V relative to those of the corresponding planar iron complexes of OEP and of the nonplanar complexes of OETPP. Such highly positive redox potentials explain the stability of these iron(II) porphyrins in air. As previously found for dodecasubstituted iron porphyrins bearing electron-withdrawing groups, the UV/Vis spectra of the[FeII(OEPy4P)(B)2]4+ complexes exhibit Soret and visible bands which are redshifted by ca. 40 nm relative to those of Fe(OEP)(Py)2. These metallo-OEPy4P4+complexes offer promising synthetic avenues to novel classes of highly charged porphyrins and multiporphyrin arrays, which are soluble in aqueous and polar organic solvents.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)