Trapping Moving Targets with Small Molecules

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159

AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.cross-reactivity | drug repositioning | cytokine | inflammation

Section: Discussionsupporting

confidence: 77%

Allosteric inhibition of macrophage migration inhibitory factor revealed by ibudilast

Cho¹,

Crichlow²,

Vermeire

et al. 2010

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159

“…To understand these complex molecules requires characterization of their free-energy landscapes-i.e., their equilibrium conformational ensembles. Precise measurements of conformational ensembles could allow quantitative modeling of the folding and function of biological macromolecules, would provide valuable experimental data to test current computational models and assumptions, and might facilitate the rational design of specifically acting inhibitors (1,2).…”

Section: Helix-junction-helix | Saxsmentioning

confidence: 99%

From a structural average to the conformational ensemble of a DNA bulge

Shi¹,

Beauchamp

Harbury

et al. 2014

Direct experimental measurements of conformational ensembles are critical for understanding macromolecular function, but traditional biophysical methods do not directly report the solution ensemble of a macromolecule. Small-angle X-ray scattering interferometry has the potential to overcome this limitation by providing the instantaneous distance distribution between pairs of gold-nanocrystal probes conjugated to a macromolecule in solution. Our X-ray interferometry experiments reveal an increasing bend angle of DNA duplexes with bulges of one, three, and five adenosine residues, consistent with previous FRET measurements, and further reveal an increasingly broad conformational ensemble with increasing bulge length. The distance distributions for the AAA bulge duplex (3A-DNA) with six different Au-Au pairs provide strong evidence against a simple elastic model in which fluctuations occur about a single conformational state. Instead, the measured distance distributions suggest a 3A-DNA ensemble with multiple conformational states predominantly across a region of conformational space with bend angles between 24 and 85 degrees and characteristic bend directions and helical twists and displacements. Additional X-ray interferometry experiments revealed perturbations to the ensemble from changes in ionic conditions and the bulge sequence, effects that can be understood in terms of electrostatic and stacking contributions to the ensemble and that demonstrate the sensitivity of X-ray interferometry. Combining X-ray interferometry ensemble data with molecular dynamics simulations gave atomic-level models of representative conformational states and of the molecular interactions that may shape the ensemble, and fluorescence measurements with 2-aminopurinesubstituted 3A-DNA provided initial tests of these atomistic models. More generally, X-ray interferometry will provide powerful benchmarks for testing and developing computational methods. helix-junction-helix | SAXSA grand challenge in biology is to understand the complex free-energy landscape of macromolecules and to decipher the resulting conformational ensembles. To perform their biological functions, macromolecules must adopt a multiplicity of conformations. Balancing and controlling different conformational states is central to biological processes including protein folding, allostery and signaling, and the stepwise assembly and function of macromolecular machines. To understand these complex molecules requires characterization of their free-energy landscapes-i.e., their equilibrium conformational ensembles. Precise measurements of conformational ensembles could allow quantitative modeling of the folding and function of biological macromolecules, would provide valuable experimental data to test current computational models and assumptions, and might facilitate the rational design of specifically acting inhibitors (1, 2).Techniques including NMR and EPR relaxation have been developed to incisively probe motions in the ensemble on different time scales, ranging from pic...

“…Identifying pockets that potentially play an allosteric role is still a major challenge but should pave the way to wide opportunities in rational drug design (23). For such purposes, we develop here an approach in which a plausible description of the conformational path between known active and inactive conformations is built and then used to identify such putative pockets.…”

mentioning

confidence: 99%

Use of allostery to identify inhibitors of calmodulin-induced activation of Bacillus anthracis edema factor

Laine

Gonçalves

Karst

et al. 2010

Allostery plays a key role in the regulation of the activity and function of many biomolecules. And although many ligands act through allostery, no systematic use is made of it in drug design strategies. Here we describe a procedure for identifying the regions of a protein that can be used to control its activity through allostery. This procedure is based on the construction of a plausible conformational path, which describes protein transition between known active and inactive conformations. The path is calculated by using a framework approach that steers and markedly improves the conjugate peak refinement method. The evolution of conformations along this path was used to identify a putative allosteric site that could regulate activation of Bacillus anthracis adenylyl cyclase toxin (EF) by calmodulin. Conformations of the allosteric site at different steps along the path from the inactive (free) to the active (bound to calmodulin) forms of EF were used to perform virtual screenings and propose candidate EF inhibitors. Several candidates then proved to inhibit calmodulin-induced activation in an in vitro assay. The most potent compound fully inhibited EF at a concentration of 10 μM. The compounds also inhibited the related adenylyl cyclase toxin from Bordetella pertussis (CyaA). The specific homology between the putative allosteric sites in both toxins supports that these pockets are the actual binding sites of the selected inhibitors.anthrax | transition path calculation | molecular dynamics | drug discovery | inhibition of protein-protein association