ortho-Aminomethylphenylboronic acid-based receptors with appended fluorophores are commonly used as molecular sensors for saccharides in aqueous media. The mechanism for fluorescence modulation in these sensors has been attributed to some form of photoinduced electron transfer (PET) quenching, which is diminished in the presence of saccharides. Using a well-known boronic acid-based saccharide sensor (3), this work reveals a new mechanism for fluorescence turn-on in these types of sensors. Compound 3 exhibits an excimer, and the associated ground-state aggregation is responsible for fluorescence modulation under certain conditions. When fructose was titrated into a solution of 3 in 2:1 water/methanol with NaCl, the fluorescence intensity increased. Yet, when the same titration was repeated in pure methanol, a solvent in which the sensor does not aggregate, no fluorescence response to fructose was observed. This reveals that the fluorescence increase is not fully associated with fructose binding, but instead disaggregation of the sensor in the presence of fructose. Further, an analogue of the sensor that does not contain a boronic acid (4) responded nearly identically to 3 in the presence of fructose, despite having no functional group with which to bind the saccharide. This further supports the claim that fluorescence modulation is not primarily a result of binding, but of disaggregation. Using an indicator displacement assay and isothermal titration calorimetry, it was confirmed that fructose does indeed bind to the sensor. Thus, our evidence reveals that while binding occurs with fructose in the aqueous solvent system used, it is not related to the majority of the fluorescence modulation. Instead, disaggregation dominates the signal turn-on, and is thus a mechanism that should be investigated in other ortho-aminomethylphenylboronic acid-based sensors.

1-Deoxy-D-xylulose 5-phosphate synthase (DXS) is a thiamin diphosphate (TDP) dependent enzyme that marks the beginning of the methylerythritol 4-phosphate isoprenoid biosynthesis pathway. The mechanism of action for DXS is still poorly understood and begins with the formation of a thiazolium ylide. This TDP activation step is thought to proceed through an intramolecular deprotonation by the 4′-amino-pyrimidine ring of TDP; however, this step would occur only after an initial deprotonation of its own 4′-amino group. The mechanism of the initial deprotonation has been hypothesized, by analogy to transketolases, to occur via a histidine or an active site water molecule. Results from hybrid quantum mechanical/molecular mechanical (QM/MM) reaction path calculations reveal an ~10 kcal/mol difference in transition state energies, favoring a water mediated mechanism over direct deprotonation by histidine. This difference was determined to be largely governed by electrostatic changes induced by conformational variations in the active site. Additionally, mutagenesis studies reveal DXS to be an evolutionarily resilient enzyme. Particularly, we hypothesize that residues H82 and H304 may act in a compensatory fashion if the other is lost due to mutation. Further, nucleus-independent chemical shifts (NICSs) and aromatic stabilization energy (ASE) calculations suggest that reduction in TDP aromaticity also serves as a factor for regulating ylide formation and controlling reactivity.

CXCL12 is a human chemokine that recognizes the CXCR4 receptor and is involved in immune responses and metastatic cancer. Interactions between CXCL12 and CXCR4 are an important drug target but, like other elongated protein-protein interfaces, present challenges for small molecule ligand discovery due to the relatively shallow and featureless binding surfaces. Calculations using an NMR complex structure revealed a binding hot spot on CXCL12 that normally interacts with the I4/I6 residues from CXCR4. Virtual screening was performed against the NMR model, and subsequent testing has verified the specific binding of multiple docking hits to this site. Together with our previous results targeting two other binding pockets that recognize sulfotyrosine residues (sY12 and sY21) of CXCR4, including a new analog against the sY12 binding site reported herein, we demonstrate that protein-protein interfaces can often possess multiple sites for engineering specific small molecule ligands that provide lead compounds for subsequent optimization by fragment based approaches.

O6-Benzylguanine is an irreversible inactivator of O6-alkylguanine-DNA alkyltransferase currently in clinical trials to overcome alkyltransferase-mediated resistance to certain cancer chemotherapeutic alkylating agents. In order to produce more soluble alkyltransferase inhibitors, we have synthesized three aminomethyl-substituted O6-benzylguanines and found that the substitution at the meta- position greatly enhances inactivation of alkyltransferase whereas para- substitution has little effect and ortho- substitution virtually eliminates activity. Molecular modeling of their interactions with alkyltransferase provided a molecular explanation for these results. The square of the correlation coefficient (R2) obtained between E-model scores (obtained from GLIDE XP/QPLD docking calculations) vs. log(ED50) values via a linear regression analysis was 0.96. The models indicate that the ortho- substitution causes a steric clash interfering with binding whereas the meta- aminomethyl substitution allows an interaction of the amino group to generate an additional hydrogen bond with the protein.