An allosteric site on MKP5 reveals a strategy for small-molecule inhibition.

The mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have been considered "undruggable," but their position as regulators of the MAPKs makes them promising therapeutic targets. MKP5 has been suggested as a potential target for the treatment of dystrophic muscle disease. Here, we identified an inhibitor of MKP5 using a p38α MAPK-derived, phosphopeptide-based small-molecule screen. We solved the structure of MKP5 in complex with this inhibitor, which revealed a previously undescribed allosteric binding pocket. Binding of the inhibitor to this pocket collapsed the MKP5 active site and was predicted to limit MAPK binding. Treatment with the inhibitor recapitulated the phenotype of MKP5 deficiency, resulting in activation of p38 MAPK and JNK. We demonstrated that MKP5 was required for TGF-ß1 signaling in muscle and that the inhibitor blocked TGF-ß1-mediated Smad2 phosphorylation. TGF-ß1 pathway antagonism has been proposed for the treatment of dystrophic muscle disease. Thus, allosteric inhibition of MKP5 represents a therapeutic strategy against dystrophic muscle disease.

An analysis of original research contributions toward FDA-approved drugs.

Academic researchers shaped the landscape of drug discovery for nearly two centuries, and their efforts initiated programs for more than half of the US Food and Drug Administration (FDA)-approved new molecular entities (NMEs). During the first 50 years of the 20th century, contributions from industry-based discovery programs steadily increased, stabilizing near half of all first publications for NMEs. Although academia and industry have made similar contributions to the discovery of FDA-approved NMEs, there remains a substantial difference in the gap-to-approval; on average, industry NMEs are 12 years closer to market at the time of the first publication. As more drug discovery efforts shift from industry to academia, including high-throughput screening resources, academia could have an increasingly crucial role in drug discovery.

A HaloTag-based small molecule microarray screening methodology with increased sensitivity and multiplex capabilities.

Small Molecule Microarrays (SMMs) represent a general platform for screening small molecule-protein interactions independent of functional inhibition of target proteins. In an effort to increase the scope and utility of SMMs, we have modified the SMM screening methodology to increase assay sensitivity and facilitate multiplex screening. Fusing target proteins to the HaloTag protein allows us to covalently prelabel fusion proteins with fluorophores, leading to increased assay sensitivity and an ability to conduct multiplex screens. We use the interaction between FKBP12 and two ligands, rapamycin and ARIAD's "bump" ligand, to show that the HaloTag-based SMM screening methodology significantly increases assay sensitivity. Additionally, using wild type FKBP12 and the FKBP12 F36V mutant, we show that prelabeling various protein isoforms with different fluorophores allows us to conduct multiplex screens and identify ligands to a specific isoform. Finally, we show this multiplex screening technique is capable of identifying ligands selective for a specific PTP1B isoform using a 20,000 compound screening deck.

Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction.

E3 ubiquitin ligases, which bind protein targets, leading to their ubiquitination and subsequent degradation, are attractive drug targets due to their exquisite substrate specificity. However, the development of small-molecule inhibitors has proven extraordinarily challenging as modulation of E3 ligase activities requires the targeting of protein-protein interactions. Using rational design, we have generated the first small molecule targeting the von Hippel-Lindau protein (VHL), the substrate recognition subunit of an E3 ligase, and an important target in cancer, chronic anemia, and ischemia. We have also obtained the crystal structure of VHL bound to our most potent inhibitor, confirming that the compound mimics the binding mode of the transcription factor HIF-1α, a substrate of VHL. These results have the potential to guide future development of improved lead compounds as therapeutics for the treatment of chronic anemia and ischemia.

Strategies for recognition of stem-loop RNA structures by synthetic ligands: application to the HIV-1 frameshift stimulatory sequence.

Production of the Gag-Pol polyprotein in human immunodeficiency virus (HIV) requires a -1 ribosomal frameshift, which is directed by a highly conserved RNA stem-loop. Building on our discovery of a set of disulfide-containing peptides that bind this RNA, we describe medicinal chemistry efforts designed to begin to understand the structure-activity relationships and RNA sequence-selectivity relationships associated with these compounds. Additionally, we have prepared analogues incorporating an olefin or saturated hydrocarbon bioisostere of the disulfide moiety, as a first step toward enhancing biostability. The olefin-containing compounds exhibit affinity comparable to the lead disulfide and, importantly, have no discernible toxicity when incubated with human fibroblasts at concentrations up to 1 mM.

Identification and characterization of a peptidic ligand for Ras.

The development of new ligands for the oncoprotein Ras can provide tools for the study of this important signaling component or potentially serve as therapeutic agents for the treatment of Ras-associated diseases. Herein, we report a peptidic Ras ligand identified through naïve phage display. Panning a phage library with a diversity of 10(9) transormants successfully identified a peptide dodecamer that contains two internal consensus motifs and binds Ras in both the active GTP- and inactive GDP-bound conformations with low micromolar dissociation constants. The dodecamer does not alter the intrinsic GTPase activity of Ras, does not compete for Ras binding to the Ras binding domain of Raf, and does not alter cell viability. This novel Ras ligand has the potential to serve in the development of higher-affinity ligands and chemical tools targeting Ras.

Allosteric site variants of Haemophilus influenzae beta-carbonic anhydrase.

Haemophilus influenzae beta-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active-site variant (D44N) that is incapable of forming a strong zinc ligand. The alteration of Trp39 to Phe increases the apparent K(i) for bicarbonate inhibition by 4.8-fold. While the structures of W39F and Y181F are very similar to the wild-type enzyme, the X-ray crystal structure of the D44N variant reveals that it has adopted an active-site conformation nearly identical to that of non-allosteric beta-carbonic anhydrases. We propose that the structure of the D44N variant is likely to be representative of the active conformation of the enzyme. These results lend additional support to the hypothesis that HICA is an allosteric enzyme that can adopt active and inactive conformations, the latter of which is stabilized by bicarbonate ion binding to a non-catalytic site.

Ribosomal frameshifting: an emerging drug target for HIV.

Combating HIV currently remains one of the key public health challenges. While immense effort has been invested in the development of HIV-targeted therapeutic agents, interfering with protein synthesis is a relatively unexplored area. HIV has an absolute requirement for a -1 ribosomal frameshift event during translation that constitutes a potentially attractive target for interfering with the viral life cycle. Research suggests that while a considerable amount of investigation is still required, targeting frameshifting in HIV with small molecules, peptides and oligonucleotides is feasible.

Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1).

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is an RNA-mediated disease. Dramatically expanded (CUG) repeats accumulate in nuclei and sequester RNA-binding proteins such as the splicing regulator MBNL1. We have employed resin-bound dynamic combinatorial chemistry (RBDCC) to identify the first examples of compounds able to inhibit MBNL1 binding to (CUG) repeat RNA. Screening an RBDCL with a theoretical diversity of 11 325 members yielded several molecules with significant selectivity for binding to (CUG) repeat RNA over other sequences. These compounds were also able to inhibit the interaction of GGG-(CUG)(109)-GGG RNA with MBNL1 in vitro, with K(i) values in the low micromolar range.

Selective recognition of alkyl pyranosides in protic and aprotic solvents.

The design and synthesis of receptors capable of selective, noncovalent recognition of carbohydrates continues to be a signature challenge in bioorganic chemistry. We report a new generation of tripodal receptors incorporating three pyridine (compound 2) or quinoline (compound 3) rings around a central cyclohexane core for use in molecular recognition of monosaccharides in apolar and polar protic solvents. These tripodal receptors were investigated using (1)H NMR, UV, and fluorescence titrations in order to determine their binding abilities toward a set of octyl glycosides. Receptor 2 displayed the highest binding affinity reported to date for noncovalent 1:1 binding of an alpha-glucopyranoside in chloroform (Ka = 212,000 +/- 27,000 M(-1)) and an approximately 8-fold selectivity for the alpha anomer over the beta anomer of the glucopyranoside. Most importantly, 2 retained its micromolar range of affinities toward monosaccharides in a polar and highly competitive solvent (methanol). The quinoline variant 3 also displayed micromolar binding affinities for selected monosaccharides in methanol (as measured by fluorescence) that were generally smaller than those of 2. Compound 3 was found to follow a selectivity pattern similar to that of 2, displaying higher affinities for glucopyranosides than for other monosaccharides. The binding stoichiometry was estimated to be 1:1 for the complexes formed by both 2 and 3 with glucopyranosides, as determined by Job plots. Nuclear Overhauser effect spectroscopy allowed for the derivation of a binding model consistent with the observed selectivities.

Synthesis and evaluation of the first cis-cyclobutane-containing receptor for lipid A.

The first example of a designed receptor containing a cis-1,3-disubstituted cyclobutane ring has been synthesized. This molecule binds diphosphoryl lipid A (a conserved portion of the Gram-(-) bacterial cell membrane, and the causative agent of septic shock) with an affinity comparable to previously described ter-cycloalkane based lipid A-binding compounds.

Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase.

The structures of beta class carbonic anhydrases (beta-CAs) determined so far fall into two distinct subclasses based on the observed coordination of the catalytic zinc (Zn2+) ion. The subclass of beta-CAs that coordinate Zn2+ tetrahedrally with four protein-derived ligands is represented by the structures of orthologues from Porphyridium purpureum, Escherichia coli, and Mycobacterium tuberculosis. Here we present the structure of an additional member of that subclass, that from Haemophilus influenzae, as well as detailed kinetic analysis, revealing the correspondence between structural classification and kinetic profile for this subclass. In addition, we identify a unique, noncatalytic binding mode for the substrate bicarbonate that occurs in both the H. influenzae and E. coli enzymes. The kinetic and structural analysis indicates that binding of bicarbonate in this site of the enzyme may modulate its activity by influencing a pH-dependent, cooperative transition between active and inactive forms. We hypothesize that the two structural subclasses of beta-CAs may provide models for the proposed active and inactive forms of the H. influenzae and E. coli enzymes.