High-Resolution Structure and Inhibition of the Schizophrenia-Linked Pseudokinase ULK4.

The ULK (UNC51-like) enzymes are a family of mammalian kinases that have critical roles in autophagy and development. While ULK1, ULK2, and ULK3 have been characterized, very little is known about ULK4. However, recently, deletions in ULK4 have been genetically linked to increased susceptibility to developing schizophrenia, a devastating neuropsychiatric disease with high heritability but few genes identified. Interestingly, ULK4 is a pseudokinase with some unusual mutations in the kinase catalytic motifs. Here, we report the first structure of the human ULK4 kinase at high resolution and show that although ULK4 has no apparent phosphotransfer activity, it can strongly bind ATP. We find an unusual mechanism for binding ATP in a Mg2+-independent manner, including a rare hydrophobic bridge in the active site. In addition, we develop two assays for ATP binding to ULK4, perform a virtual and experimental screen to identify small-molecule binders of ULK4, and identify several novel scaffolds that bind ULK4 and can lead the way to more selective small molecules that may help shed light on the function of this enigmatic protein.

A whole-animal platform to advance a clinical kinase inhibitor into new disease space.

Synthetic tailoring of approved drugs for new indications is often difficult, as the most appropriate targets may not be readily apparent, and therefore few roadmaps exist to guide chemistry. Here, we report a multidisciplinary approach for accessing novel target and chemical space starting from an FDA-approved kinase inhibitor. By combining chemical and genetic modifier screening with computational modeling, we identify distinct kinases that strongly enhance ('pro-targets') or limit ('anti-targets') whole-animal activity of the clinical kinase inhibitor sorafenib in a Drosophila medullary thyroid carcinoma (MTC) model. We demonstrate that RAF-the original intended sorafenib target-and MKNK kinases function as pharmacological liabilities because of inhibitor-induced transactivation and negative feedback, respectively. Through progressive synthetic refinement, we report a new class of 'tumor calibrated inhibitors' with unique polypharmacology and strongly improved therapeutic index in fly and human MTC xenograft models. This platform provides a rational approach to creating new high-efficacy and low-toxicity drugs.

Integrated computational and Drosophila cancer model platform captures previously unappreciated chemicals perturbing a kinase network.

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a 'hybrid' molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity.

Functional and structural analysis of rare SLC2A2 variants associated with Fanconi-Bickel syndrome and metabolic traits.

Deleterious variants in SLC2A2 cause Fanconi-Bickel Syndrome (FBS), a glycogen storage disorder, whereas less common variants in SLC2A2 associate with numerous metabolic diseases. Phenotypic heterogeneity in FBS has been observed, but its causes remain unknown. Our goal was to functionally characterize rare SLC2A2 variants found in FBS and metabolic disease-associated variants to understand the impact of these variants on GLUT2 activity and expression and establish genotype-phenotype correlations. Complementary RNA-injected Xenopus laevis oocytes were used to study mutant transporter activity and membrane expression. GLUT2 homology models were constructed for mutation analysis using GLUT1, GLUT3, and XylE as templates. Seventeen FBS variants were characterized. Only c.457_462delCTTATA (p.Leu153_Ile154del) exhibited residual glucose uptake. Functional characterization revealed that only half of the variants were expressed on the plasma membrane. Most less common variants (except c.593 C>A (p.Thr198Lys) and c.1087 G>T (p.Ala363Ser)) exhibited similar GLUT2 transport activity as the wild type. Structural analysis of GLUT2 revealed that variants affect substrate-binding, steric hindrance, or overall transporter structure. The mutant transporter that is associated with a milder FBS phenotype, p.Leu153_Ile154del, retained transport activity. These results improve our overall understanding of the underlying causes of FBS and impact of GLUT2 function on various clinical phenotypes ranging from rare to common disease.

Identifying binding hot spots on protein surfaces by mixed-solvent molecular dynamics: HIV-1 protease as a test case.

Mixed-solvent molecular dynamics (MixMD) simulations use full protein flexibility and competition between water and small organic probes to achieve accurate hot-spot mapping on protein surfaces. In this study, we improved MixMD using human immunodeficiency virus type-1 protease (HIVp) as the test case. We used three probe-water solutions (acetonitrile-water, isopropanol-water, and pyrimidine-water), first at 50% w/w concentration and later at 5% v/v. Paradoxically, better mapping was achieved by using fewer probes; 5% simulations gave a superior signal-to-noise ratio and far fewer spurious hot spots than 50% MixMD. Furthermore, very intense and well-defined probe occupancies were observed in the catalytic site and potential allosteric sites that have been confirmed experimentally. The Eye site, an allosteric site underneath the flap of HIVp, has been confirmed by the presence of a 5-nitroindole fragment in a crystal structure. MixMD also mapped two additional hot spots: the Exo site (between the Gly16-Gly17 and Cys67-Gly68 loops) and the Face site (between Glu21-Ala22 and Val84-Ile85 loops). The Exo site was observed to overlap with crystallographic additives such as acetate and dimethyl sulfoxide that are present in different crystal forms of the protein. Analysis of crystal structures of HIVp in different symmetry groups has shown that some surface sites are common interfaces for crystal contacts, which means that they are surfaces that are relatively easy to desolvate and complement with organic molecules. MixMD should identify these sites; in fact, their occupancy values help establish a solid cut-off where "druggable" sites are required to have higher occupancies than the crystal-packing faces.

Discovery and Optimization of Selective Brain-Penetrant EBP Inhibitors that Enhance Oligodendrocyte Formation.

The inhibition of emopamil binding protein (EBP), a sterol isomerase within the cholesterol biosynthesis pathway, promotes oligodendrocyte formation, which has been proposed as a potential therapeutic approach for treating multiple sclerosis. Herein, we describe the discovery and optimization of brain-penetrant, orally bioavailable inhibitors of EBP. A structure-based drug design approach from literature compound 1 led to the discovery of a hydantoin-based scaffold, which provided balanced physicochemical properties and potency and an improved in vitro safety profile. The long half-lives of early hydantoin-based EBP inhibitors in rodents prompted an unconventional optimization strategy, focused on increasing metabolic turnover while maintaining potency and a brain-penetrant profile. The resulting EBP inhibitor 11 demonstrated strong in vivo target engagement in the brain, as illustrated by the accumulation of EBP substrate zymostenol after repeated dosing. Furthermore, compound 11 enhanced the formation of oligodendrocytes in human cortical organoids, providing additional support for our therapeutic hypothesis.

Exploiting Allosteric Properties of RAF and MEK Inhibitors to Target Therapy-Resistant Tumors Driven by Oncogenic BRAF Signaling.

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF-MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy-resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors. SIGNIFICANCE: This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors.See related commentary by Zhang and Bollag, p. 1620.This article is highlighted in the In This Issue feature, p. 1601.

Chemical probes that selectively recognize the earliest Aß oligomers in complex mixtures.

Alzheimer's disease (AD) is characterized by the self-assembly of amyloid beta (Aß) peptides. Recent models implicate some of the earliest Aß oligomers, such as trimers and tetramers, in disease. However, the roles of these structures remain uncertain, in part, because selective probes of their formation are not available. Toward that goal, we generated bivalent versions of the known Aß ligand, the pentapeptide KLVFF. We found that compounds containing sufficiently long linkers (∼19 to 24 Å) recognized primarily Aß trimers and tetramers, with little binding to either monomer or higher order structures. These compounds might be useful probes for early Aß oligomers.

Automated genome mining for natural products.

BACKGROUND: Discovery of new medicinal agents from natural sources has largely been an adventitious process based on screening of plant and microbial extracts combined with bioassay-guided identification and natural product structure elucidation. Increasingly rapid and more cost-effective genome sequencing technologies coupled with advanced computational power have converged to transform this trend toward a more rational and predictive pursuit. RESULTS: We have developed a rapid method of scanning genome sequences for multiple polyketide, nonribosomal peptide, and mixed combination natural products with output in a text format that can be readily converted to two and three dimensional structures using conventional software. Our open-source and web-based program can assemble various small molecules composed of twenty standard amino acids and twenty two other chain-elongation intermediates used in nonribosomal peptide systems, and four acyl-CoA extender units incorporated into polyketides by reading a hidden Markov model of DNA. This process evaluates and selects the substrate specificities along the assembly line of nonribosomal synthetases and modular polyketide synthases. CONCLUSION: Using this approach we have predicted the structures of natural products from a diverse range of bacteria based on a limited number of signature sequences. In accelerating direct DNA to metabolomic analysis, this method bridges the interface between chemists and biologists and enables rapid scanning for compounds with potential therapeutic value.

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.

Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism.

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.

A poke in the eye: inhibiting HIV-1 protease through its flap-recognition pocket.

A novel mechanism of inhibiting HIV-1 protease (HIVp) is presented. Using computational solvent mapping to identify complementary interactions and the Multiple Protein Structure method to incorporate protein flexibility, we generated a receptor-based pharmacophore model of the flexible flap region of the semiopen, apo state of HIVp. Complementary interactions were consistently observed at the base of the flap, only within a cleft with a specific structural role. In the closed, bound state of HIVp, each flap tip docks against the opposite monomer, occupying this cleft. This flap-recognition site is filled by the protein and cannot be identified using traditional approaches based on bound, closed structures. Virtual screening and dynamics simulations show how small molecules can be identified to complement this cleft. Subsequent experimental testing confirms inhibitory activity of this new class of inhibitor. This may be the first new inhibitor class for HIVp since dimerization inhibitors were introduced 17 years ago.

Role of the CCAAT/enhancer-binding protein NFATc2 transcription factor cascade in the induction of secretory phospholipase A2.

Inflammatory cytokines such as interleukin-1 and tumor necrosis factor-alpha modulate a transcription factor cascade in the liver to induce and sustain an acute and systemic defense against foreign entities. The transcription factors involved include NF-kappaB, STAT, and CCAAT/enhancer-binding protein (C/EBP). Whether the NFAT group of transcription factors (which was first characterized as playing an important role in cytokine gene expression in the adaptive response in immune cells) participates in the acute-phase response in hepatocytes is not known. Here, we have investigated whether NFAT is part of the transcription factor cascade in hepatocytes during inflammatory stress. We report that interleukin-1 or tumor necrosis factor-alpha increases expression of and activates NFATc2. C/EBP-mediated NFATc2 induction is temporally required for expression of type IIA secretory phospholipase A2. NFATc2 is also required for expression of phospholipase D1 and the calcium-binding protein S100A3. Thus, a C/EBP-NFATc2 transcription factor cascade provides an additional means to modulate the acute-phase response upon stimulation with inflammatory cytokines.