High-resolution crystal structure and chemical screening reveal pantothenate kinase as a new target for antifungal development.

Fungal infections are the leading cause of mortality by eukaryotic pathogens, with an estimated 150 million severe life-threatening cases and 1.7 million deaths reported annually. The rapid emergence of multidrug-resistant fungal isolates highlights the urgent need for new drugs with new mechanisms of action. In fungi, pantothenate phosphorylation, catalyzed by PanK enzyme, is the first step in the utilization of pantothenic acid and coenzyme A biosynthesis. In all fungi sequenced so far, this enzyme is encoded by a single PanK gene. Here, we report the crystal structure of a fungal PanK alone as well as with high-affinity inhibitors from a single chemotype identified through a high-throughput chemical screen. Structural, biochemical, and functional analyses revealed mechanisms governing substrate and ligand binding, dimerization, and catalysis and helped identify new compounds that inhibit the growth of several Candida species. The data validate PanK as a promising target for antifungal drug development.

Phosphorylated WNK kinase networks in recoded bacteria recapitulate physiological function.

Advances in genetic code expansion have enabled the production of proteins containing site-specific, authentic post-translational modifications. Here, we use a recoded bacterial strain with an expanded genetic code to encode phosphoserine into a human kinase protein. We directly encode phosphoserine into WNK1 (with-no-lysine [K] 1) or WNK4 kinases at multiple, distinct sites, which produced activated, phosphorylated WNK that phosphorylated and activated SPAK/OSR kinases, thereby synthetically activating this human kinase network in recoded bacteria. We used this approach to identify biochemical properties of WNK kinases, a motif for SPAK substrates, and small-molecule kinase inhibitors for phosphorylated SPAK. We show that the kinase inhibitors modulate SPAK substrates in cells, alter cell volume, and reduce migration of glioblastoma cells. Our work establishes a protein-engineering platform technology that demonstrates that synthetically active WNK kinase networks can accurately model cellular systems and can be used more broadly to target networks of phosphorylated proteins for research and discovery.

Targeting Acidic Mammalian chitinase Is Effective in Animal Model of Asthma.

This article highlights our work toward the identification of a potent, selective, and efficacious acidic mammalian chitinase (AMCase) inhibitor. Rational design, guided by X-ray analysis of several inhibitors bound to human chitotriosidase (hCHIT1), led to the identification of compound 7f as a highly potent AMCase inhibitor (IC50 values of 14 and 19 nM against human and mouse enzyme, respectively) and selective (>150× against mCHIT1) with very good PK properties. This compound dosed once daily at 30 mg/kg po showed significant anti-inflammatory efficacy in HDM-induced allergic airway inflammation in mice, reducing inflammatory cell influx in the BALF and total IgE concentration in plasma, which correlated with decrease of chitinolytic activity. Therapeutic efficacy of compound 7f in the clinically relevant aeroallergen-induced acute asthma model in mice provides a rationale for developing AMCase inhibitor for the treatment of asthma.

Pyrrolocin A, a 3-Decalinoyltetramic Acid with Selective Biological Activity, Isolated from Amazonian Cultures of the Novel Endophyte Diaporthales sp. E6927E.

Natural products remain an important source of new therapeutics for emerging drug-resistant pathogens like Candida albicans, which particularly affects immunocompromised patients. A bioactive 3-decalinoyltetramic acid, pyrrolocin A, was isolated from extracts of a novel Amazonian fungal endophyte, E6927E, of the Diaporthales family. The structure of the natural product was solved using NMR and CD spectroscopy and it is structurally related to the fungal setins, equisetin and phomasetin, which are well-characterized tetramic acid antibiotics specific for Gram-positive organisms. We show that the compound inhibits growth of Staphylococcus aureus and Enterococcus faecalis. It shows selective and potent bioactivity against fungal strains, with an MIC of 4 µg/mL for C. albicans, 100 µg/mL for Aspergillus sp. and greater than 100 µg/mL for Saccharomyces cerevisiae. Further, the compound is less toxic to mammalian cells (IC50 = 150 µg/mL), with an inhibitory concentration greater than forty times that for C. albicans. Pyrrolocin A retained potent activity against eight out of seventeen strains of clinical Candida sp. isolates tested.

YU238259 Is a Novel Inhibitor of Homology-Dependent DNA Repair That Exhibits Synthetic Lethality and Radiosensitization in Repair-Deficient Tumors.

UNLABELLED: Radiotherapy and DNA-damaging chemotherapy are frequently utilized in the treatment of solid tumors. Innate or acquired resistance to these therapies remains a major clinical challenge in oncology. The development of small molecules that sensitize cancers to established therapies represents an attractive approach to extending survival and quality of life in patients. Here, we demonstrate that YU238259, a member of a novel class of DNA double-strand break repair inhibitors, exhibits potent synthetic lethality in the setting of DNA damage response and DNA repair defects. YU238259 specifically inhibits homology-dependent DNA repair, but not non-homologous end-joining, in cell-based GFP reporter assays. Treatment with YU238259 is not only synergistic with ionizing radiation, etoposide, and PARP inhibition, but this synergism is heightened by BRCA2 deficiency. Further, growth of BRCA2-deficient human tumor xenografts in nude mice is significantly delayed by YU238259 treatment even in the absence of concomitant DNA-damaging therapy. The cytotoxicity of these small molecules in repair-deficient cells results from an accumulation of unresolved DNA double-strand breaks. These findings suggest that YU238259 or related small molecules may have clinical benefit to patients with advanced BRCA2-negative tumors, either as a monotherapy or as an adjuvant to radiotherapy and certain chemotherapies. IMPLICATIONS: We have identified a novel series of compounds that demonstrate synthetic lethality in DNA repair-deficient cell and animal models and have strong potential for clinical translation.

Discovery and synthesis of novel 4-aminopyrrolopyrimidine Tie-2 kinase inhibitors for the treatment of solid tumors.

The synthesis and biological evaluation of novel Tie-2 kinase inhibitors are presented. Based on the pyrrolopyrimidine chemotype, several new series are described, including the benzimidazole series by linking a benzimidazole to the C5-position of the 4-amino-pyrrolopyrimidine core and the ketophenyl series synthesized by incorporating a ketophenyl group to the C5-position. Medicinal chemistry efforts led to potent Tie-2 inhibitors. Compound 15, a ketophenyl pyrrolopyrimidine urea analog with improved physicochemical properties, demonstrated favorable in vitro attributes as well as dose responsive and robust oral tumor growth inhibition in animal models.

Short-acting 5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one derivatives as orally-active calcium-sensing receptor antagonists.

Synthesis and structure-activity relationship (SAR) studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones, a novel class of calcium receptor antagonists is described with particular emphasis on optimization of the pharmacokinetic/pharmacodynamic parameters required for a short duration of action compound. Orally-active compounds were identified which displayed the desired animal pharmacology (rapid and transient stimulation of parathyroid hormone) essential for bone anabolic effects.

The discovery of highly selective erbB2 (Her2) inhibitors for the treatment of cancer.

The synthesis and biological evaluation of potent and selective inhibitors of the erbB2 kinase is presented. Based on the 4-anilinoquinazoline chemotype, the syntheses of several new series of erbB2 inhibitors are described with quinazoline and pyrido[4,3-d]pyrimidine cores. The vast majority of these compounds are found to be >100x selective over the closely related EGFR kinase. Two lead compounds are further shown to have low clearance and moderate bioavailability in rat.