Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl‑1

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein–protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein–protein interactions

Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl‑1

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein–protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein–protein interactions

Mixture-Based Screening of Focused Combinatorial Libraries by NMR: Application to the Antiapoptotic Protein hMcl‑1

We report on an innovative ligand discovery strategy based on protein NMR-based screening of a combinatorial library of ∼125,000 compounds that was arranged in 96 distinct mixtures. Using sensitive solution protein NMR spectroscopy and chemical perturbation-based screening followed by an iterative synthesis, deconvolutions, and optimization strategy, we demonstrate that the approach could be useful in the identification of initial binding molecules for difficult drug targets, such as those involved in protein–protein interactions. As an application, we will report novel agents targeting the Bcl-2 family protein hMcl-1. The approach is of general applicability and could be deployed as an effective screening strategy for de novo identification of ligands, particularly when tackling targets involved in protein–protein interactions

Enthalpy-Based Screening of Focused Combinatorial Libraries for the Identification of Potent and Selective Ligands

In modern drug discovery, the ability of biophysical methods, including nuclear magnetic resonance spectroscopy or surface plasmon resonance, to detect and characterize ligand–protein interactions accurately and unambiguously makes these approaches preferred versus conventional biochemical high-throughput screening of large collections of compounds. Nonetheless, ligand screening strategies that address simultaneously potency and selectivity have not yet been fully developed. In this work, we propose a novel method for screening large collections of combinatorial libraries using enthalpy measurements as a primary screening technique. We demonstrate that selecting binders that are driven by enthalpy (Δ<i>H</i>) results in agents that are not only potent but also more selective for a given target. This general and novel approach, we termed Δ<i>H</i> screening of <i>f</i>POS (enthalpy screening of focused positional scanning library), combines the principles of focused combinatorial chemistry with rapid calorimetry measurements to efficiently identify potent and selective inhibitors

HTS by NMR for the Identification of Potent and Selective Inhibitors of Metalloenzymes

We have recently proposed a novel drug discovery approach based on biophysical screening of focused positional scanning libraries in which each element of the library contained a common binding moiety for the given target or class of targets. In this Letter, we report on the implementation of this approach to target metal containing proteins. In our implementation, we first derived a focused positional scanning combinatorial library of peptide mimetics (of approximately 100,000 compounds) in which each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal. Screening of this library by nuclear magnetic resonance spectroscopy in solution allowed the identification of a novel and selective compound series targeting MMP-12. The data supported that our general approach, perhaps applied using other metal chelating agents or other initial binding fragments, may result very effective in deriving novel and selective agents against metalloenzyme

HTS by NMR for the Identification of Potent and Selective Inhibitors of Metalloenzymes

We have recently proposed a novel drug discovery approach based on biophysical screening of focused positional scanning libraries in which each element of the library contained a common binding moiety for the given target or class of targets. In this Letter, we report on the implementation of this approach to target metal containing proteins. In our implementation, we first derived a focused positional scanning combinatorial library of peptide mimetics (of approximately 100,000 compounds) in which each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal. Screening of this library by nuclear magnetic resonance spectroscopy in solution allowed the identification of a novel and selective compound series targeting MMP-12. The data supported that our general approach, perhaps applied using other metal chelating agents or other initial binding fragments, may result very effective in deriving novel and selective agents against metalloenzyme

Design of Potent pan-IAP and Lys-Covalent XIAP Selective Inhibitors Using a Thermodynamics Driven Approach

Recently we reported that rapid determination of enthalpy of binding can be achieved for a large number of congeneric agents or in combinatorial libraries fairly efficiently. We show that using a thermodynamic Craig plot can be very useful in dissecting the enthalpy and entropy contribution of different substituents on a common scaffold, in order to design potent, selective, or pan-active compounds. In our implementation, the approach identified a critical Lys residue in the BIR3 domain of XIAP. We report for the first time that it is possible to target such residue covalently to attain potent and selective agents. Preliminary cellular studies in various models of leukemia, multiple myeloma, and pancreatic cancers suggest that the derived agents possess a potentially intriguing pattern of activity, especially for cell lines that are resistant to the pan-IAP antagonist and clinical candidate LCL161

Endoplasmic reticulum membrane reorganization is regulated by ionic homeostasis.

Recently we described a new, evolutionarily conserved cellular stress response characterized by a reversible reorganization of endoplasmic reticulum (ER) membranes that is distinct from canonical ER stress and the unfolded protein response (UPR). Apogossypol, a putative broad spectrum BCL-2 family antagonist, was the prototype compound used to induce this ER membrane reorganization. Following microarray analysis of cells treated with apogossypol, we used connectivity mapping to identify a wide range of structurally diverse chemicals from different pharmacological classes and established their ability to induce ER membrane reorganization. Such structural diversity suggests that the mechanisms initiating ER membrane reorganization are also diverse and a major objective of the present study was to identify potentially common features of these mechanisms. In order to explore this, we used hierarchical clustering of transcription profiles for a number of chemicals that induce membrane reorganization and discovered two distinct clusters. One cluster contained chemicals with known effects on Ca(2+) homeostasis. Support for this was provided by the findings that ER membrane reorganization was induced by agents that either deplete ER Ca(2+) (thapsigargin) or cause an alteration in cellular Ca(2+) handling (calmodulin antagonists). Furthermore, overexpression of the ER luminal Ca(2+) sensor, STIM1, also evoked ER membrane reorganization. Although perturbation of Ca(2+) homeostasis was clearly one mechanism by which some agents induced ER membrane reorganization, influx of extracellular Na(+) but not Ca(2+) was required for ER membrane reorganization induced by apogossypol and the related BCL-2 family antagonist, TW37, in both human and yeast cells. Not only is this novel, non-canonical ER stress response evolutionary conserved but so also are aspects of the mechanism of formation of ER membrane aggregates. Thus perturbation of ionic homeostasis is important in the regulation of ER membrane reorganization

Design of Potent pan-IAP and Lys-Covalent XIAP Selective Inhibitors Using a Thermodynamics Driven Approach

Recently we reported that rapid determination of enthalpy of binding can be achieved for a large number of congeneric agents or in combinatorial libraries fairly efficiently. We show that using a thermodynamic Craig plot can be very useful in dissecting the enthalpy and entropy contribution of different substituents on a common scaffold, in order to design potent, selective, or pan-active compounds. In our implementation, the approach identified a critical Lys residue in the BIR3 domain of XIAP. We report for the first time that it is possible to target such residue covalently to attain potent and selective agents. Preliminary cellular studies in various models of leukemia, multiple myeloma, and pancreatic cancers suggest that the derived agents possess a potentially intriguing pattern of activity, especially for cell lines that are resistant to the pan-IAP antagonist and clinical candidate LCL161

ER membrane reorganization may be due to enhanced Ca²⁺ entry.

<p>(A) Fura-2-loaded HeLa cells were exposed to either apogossypol (10 µM) or THG (10 µM) in Ca²⁺-free media. This was followed by addition of extracellular Ca²⁺. Changes in fura-2 fluorescence were measured throughout as an index of changes in [Ca²⁺]_cyt. The graph represents the mean from 3 independent experiments with 20 cells recorded in each experiment. (B) Extensive ER membrane reorganization, assessed by BAP31 staining, was observed in HeLa cells exposed for 4 h to the calmodulin inhibitors, calmidazolium (10 µM) and A-7 (20 µM) (scale bar, 20 µm). (C) Apogossypol-mediated ER membrane reorganization was abolished when HeLa cells were pretreated for 1 h with 2-APB (10 µM) (scale bar, 20 µm). (D) Transient overexpression of YFP, STIM1-YFP, ORAI1-YFP, ORAI2-Myc or ORAI3-Myc and immunostaining with BAP31 antibody, revealed that STIM1-YFP caused extensive ER membrane reorganization, similar to that observed following apogossypol (scale bar, 10 µm). (E) Ultrastructure of STIM1-YFP expressing HeLa cells showing STIM1-mediated reorganization of ER membranes (lower two panels). The bottom panel (scale bar, 5 µm) shows a detail of one of two regions of pronounced ER membrane organizations (indicated by black arrows in the middle panel). It also shows a clear continuity of membranes with the rest of the ER (white arrows). The upper panel shows a comparable area of control transfected HeLa cell (the scale bar in the top 2 panels is 2 µm).</p