Enantioselective Organocatalytic Hantzsch Synthesis of Polyhydroquinolines

The four-component Hantzsch reaction provides access to pharmaceutically important dihydropyridines. To expand the utility of this method, we have developed a route under organocatalytic conditions with good yields and excellent ee’s. Through catalyst screening, we found that a BINOL−phosphoric acid allowed enantioselective synthesis of six-membered heterocycles with a variety of substitution patterns

Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation

Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone–TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein–protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, <i>in vitro</i>. These studies identified some surprising selectivity among the chaperone–TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones

Control of Multivalent Interactions by Binding Epitope Density

Receptor clustering by multivalent ligands can activate signaling pathways. In principle, multivalent ligand features can control clustering and the downstream signals that result, but the influence of ligand structure on these processes is incompletely understood. Using a series of synthetic polymers that vary systematically, we studied the influence of multivalent ligand binding epitope density on the clustering of a model receptor, concanavalin A (Con A). We analyze three aspects of receptor clustering:  the stoichiometry of the complex, rate of cluster formation, and receptor proximity. Our experiments reveal that the density of binding sites on a multivalent ligand strongly influences each of these parameters. In general, high binding epitope density results in greater numbers of receptors bound per polymer, faster rates of clustering, and reduced inter-receptor distances. Ligands with low binding epitope density, however, are the most efficient on a binding epitope basis. Our results provide insight into the design of ligands for controlling receptor−receptor interactions and can be used to illuminate mechanisms by which natural multivalent displays function

Selective Targeting of Cells via Bispecific Molecules That Exploit Coexpression of Two Intracellular Proteins

In drug discovery, small molecules must often discriminate between healthy and diseased cells. This feat is usually accomplished by binding to a protein that is preferentially expressed in the target cell or on its surface. However, in many cases, the expression of an individual protein may not generate sufficient cyto-selectivity. Here, we demonstrate that bispecific molecules can better discriminate between similar cell types by exploiting their simultaneous affinity for two proteins. Inspired by the natural product FK506, we designed molecules that have affinity for both FKBP12 and HIV protease. Using cell-based reporters and live virus assays, we observed that these compounds preferentially accumulated in cells that express both targets, mimicking an infected lymphocyte. Treatment with FKBP12 inhibitors reversed this partitioning, while overexpression of FKBP12 protein further promoted it. The partitioning into the target cell type could be tuned by controlling the properties of the linker and the affinities for the two proteins. These results show that bispecific molecules create significantly better potential for cyto-selectivity, which might be especially important in the development of safe and effective antivirals and anticancer compounds

Synthesis and Applications of End-Labeled Neoglycopolymers

Neoglycopolymers that vary in length and contain a single fluorescent reporter group were synthesized using ring-opening metathesis polymerization (ROMP). The utility of these materials is demonstrated by the development of a cellular binding assay for L-selectin, a cell surface protein that plays a role in inflammation. The data reveal that these multivalent ligands interact with multiple copies of L-selectin

Designing de Novo Small Molecules That Control Heat Shock Protein 70 (Hsp70) and Heat Shock Organizing Protein (HOP) within the Chaperone Protein-Folding Machinery

Protein–protein interactions (PPIs) regulate all signaling pathways for cellular function. Developing molecules that modulate PPIs through the interface of their protein surfaces has been a significant challenge and there has been little success controlling PPIs through standard molecular library screening approaches. PPIs control the cell’s protein-folding machinery, and this machinery relies on a multiprotein complex formed with heat shock protein 70 (Hsp70). Described is the design, synthesis, and biological evaluation of molecules aimed to regulate the interaction between two proteins that are critical to the protein-folding machinery: heat shock protein 70 (Hsp70) and cochaperone heat shock organizing protein (HOP). We report the first class of compounds that directly regulate these two protein–protein interactions and inhibit protein folding events

An Allosteric Modulator of HIV‑1 Protease Shows Equipotent Inhibition of Wild-Type and Drug-Resistant Proteases

NMR and MD simulations have demonstrated that the flaps of HIV-1 protease (HIV-1p) adopt a range of conformations that are coupled with its enzymatic activity. Previously, a model was created for an allosteric site located between the flap and the core of HIV-1p, called the Eye site (Biopolymers 2008, 89, 643−652). Here, results from our first study were combined with a ligand-based, lead-hopping method to identify a novel compound (NIT). NIT inhibits HIV-1p, independent of the presence of an active-site inhibitor such as pepstatin A. Assays showed that NIT acts on an allosteric site other than the dimerization interface. MD simulations of the ligand–protein complex show that NIT stably binds in the Eye site and restricts the flaps. That bound state of NIT is consistent with a crystal structure of similar fragments bound in the Eye site (Chem. Biol. Drug Des. 2010, 75, 257−268). Most importantly, NIT is equally potent against wild-type and a multidrug-resistant mutant of HIV-1p, which highlights the promise of allosteric inhibitors circumventing existing clinical resistance

Examples of Ramachandran plots of NBD residues in the LD simulations.

<p>(A) Density maps of the φ-ψ torsion angles that were highly occupied (red). Residues not involved in hinged movement usually had one φ-ψ torsion angle cluster in the plot (A191 and K245). However, other “hinge” residues had two or more φ-ψ clusters (G74 and G228), which indicated substantial conformational changes in the backbone. (B) Comparing the ψ-φ clusters generated from the ADP/P_i-bound (red) and ATP-bound (black) trajectories. Residues not involved in hinged movement (A191, K245) have overlapped φ-ψ clusters, while hinge residues have φ-ψ clusters that differ significantly. Some of these hinge residues were affected by nucleotide changes (G228), while others were not (G74). (C) Cartoon representation of Domain I, in which residues have multiple Cα φ-ψ states during dynamics simulations are shown. Subdomain I-A is colored in gray and I-B in white. (D) Cartoon representation of Domain II. Subdomain II-A is colored in gray and II-B in white. Residues with multiple φ-ψ states are colored in red. Most colored residues are located in random coils, loops or end of the α-helices or β-sheets.</p

Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein–Protein Interactions

Methods for identifying chemical inhibitors of protein–protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70–Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods

The motions of the nucleotides are correlated with several residues.

<p>(A) Residues correlated with the nucleotides, listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003279#pcbi-1003279-t003" target="_blank">Table 3</a>, are colored by how frequently the behavior is observed in the LD simulations of ATP- and ADP/P_i-bound NBD: red, 10 out of 10; yellow, 6 out of 10; green, 4 out of 10; blue <4. (B) Most of the correlated residues are in the nucleotide binding site; here, we highlight the residues with multiple φ-ψ torsion angle clusters, strong correlated motion to nucleotides, and >8 Å away from nucleotide.</p