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[Image: see text] The selective inhibition of RET kinase as a treatment for relevant cancer types including lung adenocarcinoma has garnered considerable interest in recent years and prompted a variety of efforts toward the discovery of small-molecule therapeutics. Hits uncovered via the analysis of archival kinase data ultimately led to the identification of a promising pyrrolo[2,3-d]pyrimidine scaffold. The optimization of this pyrrolo[2,3-d]pyrimidine core resulted in compound 1, which demonstrated potent in vitro RET kinase inhibition and robust in vivo efficacy in RET-driven tumor xenografts upon multiday dosing in mice. The administration of 1 was well-tolerated at established efficacious doses (10 and 30 mg/kg, po, qd), and plasma exposure levels indicated a minimal risk of KDR or hERG inhibition in vivo, as evaluated by Miles assay and free plasma concentrations, respectively.

Visceral leishmaniasis is responsible for up to 30,000 deaths every year. Current treatments have shortcomings that include toxicity and variable efficacy across endemic regions. Previously, we reported the discovery of GNF6702, a selective inhibitor of the kinetoplastid proteasome, which cleared parasites in murine models of leishmaniasis, Chagas disease, and human African trypanosomiasis. Here, we describe the discovery and characterization of LXE408, a structurally related kinetoplastid-selective proteasome inhibitor currently in Phase 1 human clinical trials. Furthermore, we present high-resolution cryo-EM structures of the Leishmania tarentolae proteasome in complex with LXE408, which provides a compelling explanation for the noncompetitive mode of binding of this novel class of inhibitors of the kinetoplastid proteasome.

RET (REarranged during Transfection) kinase gain-of-function aberrancies have been identified as potential oncogenic drivers in lung adenocarcinoma, along with several other cancer types, prompting the discovery and assessment of selective inhibitors. Internal mining and analysis of relevant kinase data informed the decision to investigate a pyrazolo[1,5-a]pyrimidine scaffold, where subsequent optimization led to the identification of compound WF-47-JS03 (1), a potent RET kinase inhibitor with >500-fold selectivity against KDR (Kinase insert Domain Receptor) in cellular assays. In subsequent mouse in vivo studies, compound 1 demonstrated effective brain penetration and was found to induce strong regression of RET-driven tumor xenografts at a well-tolerated dose (10 mg/kg, po, qd). Higher doses of 1, however, were poorly tolerated in mice, similar to other pyrazolo[1,5-a]pyrimidine compounds at or near the efficacious dose, and indicative of the narrow therapeutic windows seen with this scaffold.

NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually1. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target2. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Legumes establish mutualistic associations with nitrogen-fixing bacteria, commonly referred to as rhizobia. Most rhizobia enter roots of host plants via root hairs, where they induce the formation of infection threads. Bacterial invasion and formation of nodules containing nitrogen-fixing bacteroids (the Fix+ phenotype) depend on a molecular dialogue between the symbiotic partners. Host plants secrete flavonoids that induce the synthesis of rhizobial lipo-chito-oligosaccharides called Nod factors (24, 26) and perceive them via a specific signal transduction pathway that culminates in expression of symbiosis-specific plant genes required for nodule development (34). In many symbiotic associations, nodule formation also depends on rhizobial macromolecules that include exopolysaccharides (EPS), lipopolysaccharides, K-antigens, and cyclic β-glucans. These extracellular polysaccharides (or the oligosaccharides derived from them) are crucial in the early stages of nodule formation that begins after rhizobia have entered the host plant (3, 8, 17, 19, 25, 33, 36). In addition to a protective role, rhizobial EPS have vital but poorly understood roles in infection of legume roots (2). The EPS structures of various rhizobia, including Rhizobium sp. strain NGR234 (also called Sinorhizobium fredii NGR234), which nodulates more than 112 genera of legumes (30), have been described. The repeating subunit of the acidic EPS of NGR234 is a nonasaccharide consisting of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues (9) (Fig. ​(Fig.1A).1A). Mutants of NGR234 deficient in EPS synthesis formed nonmucoid (“dry”) colonies on agar plates and lost the capacity to induce the formation of nitrogen-fixing nodules on Leucaena leucocephala (Fix− phenotype). Instead, small empty nodules (pseudonodules) were formed (5). A number of genes of NGR234 involved in synthesis of EPS have been identified in an exo cluster (6, 15, 45), which has been sequenced (38) (Fig. ​(Fig.1B).1B). This cluster is located on the pNGR234b megaplasmid and is similar to the exo cluster of Sinorhizobium meliloti strain 1021 that is required for synthesis of succinoglycan (also called EPS I) (13). Based on known or assumed functions of exo genes in S. meliloti (2, 32), we predict that EPS synthesis in NGR234 depends on the glycosyl transferases (ExoY, ExoA, ExoL, ExoM, ExoO, and ExoU) that form an undecaprenol diphosphate-linked repeating subunit at the cytoplasmic face of the inner membrane. Probably, the lipid-linked subunits are then flipped across the inner membrane and finally assembled by an ExoQ/ExoP/ExoF-dependent polymerization system that is similar to the Wzy polymerization pathway of group 1 capsular polysaccharides in Escherichia coli (41). In this model, ExoQ is a putative polymerase and ExoP is a copolymerase belonging to the MPA1 (cytoplasmic membrane-periplasmic auxiliary protein 1) family. ExoF is probably an outer membrane auxiliary protein required for EPS export. Related genes required for secretion and export of EPS (pssL, pssT, pssP, and pssN) have been identified in Rhizobium leguminosarum bv. trifolii (reference 22 and references therein). FIG. 1. (A) Repeating subunit of the acidic EPS from NGR234 (9). Additional acetyl groups are present at unidentified positions. (B) Genetic map of the exo cluster of pNGR234b (38). The exs genes and neighboring exoB gene are not shown. BamHI fragments were cloned ... Low-molecular-weight (LMW) forms of EPS (called exo-oligosaccharides [EOSs]) have been identified in supernatants of rhizobial cultures. Nodulation experiments with purified LMW EPS applied to roots indicated that EOSs could complement the symbiotic defects of exo mutants under certain growth conditions (1, 10, 14, 40). Similar complementation effects were observed when exo mutants were coinoculated with noninvasive strains that synthesize EPS (17, 18). Although not always conclusive, the results of such complementation experiments suggest that EOSs are symbiotically active molecules in certain plants. Synthesis of EOSs seems to be controlled either by enzymes involved in export and polymerization of the lipid-linked repeating EPS subunit or by extracellular glycanases that release EOSs from high-molecular-weight (HMW) forms (14). Glycanases that cleave EPS have been identified in S. meliloti and R. leguminosarum bv. viciae. Corresponding mutants produced lower levels of EOSs without altering the symbiotic phenotype (12, 43). The previously characterized glycanase ExoK secreted by S. meliloti (43, 44) exhibits a high level of sequence similarity [e−129] to the putative ExoK protein of NGR234. In this study, we mutated exo genes of NGR234 involved in synthesis of the repeating subunit (exoL, exoY, and exoZ), polymerization (exoF, exoP, and exoQ), and production of EOSs (exoK). We provide evidence that EOSs of NGR234 are required for symbiosis with certain plants.

Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum , RBL5808 and RBL5812, revealed independent Tn 5 transposon integrations in a single gene, designated exo5 . As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.

Treatment options for androgen-independent prostate cancer cells are limited. Therefore, it is critical to identify agents that induce death of both androgen-responsive and androgen-insensitive cells. Here we demonstrate that a product of plant cell walls, pectin, is capable of inducing apoptosis in androgen-responsive (LNCaP) and androgen-independent (LNCaP C4-2) human prostate cancer cells. Commercially available fractionated pectin powder (FPP) induced apoptosis (approximately 40-fold above non-treated cells) in both cell lines as determined by the Apoptosense assay and activation of caspase-3 and its substrate, poly(ADP-ribose) polymerase. Conversely, citrus pectin (CP) and the pH-modified CP, PectaSol, had little or no apoptotic activity. Glycosyl residue composition and linkage analyses revealed no significant differences among the pectins. Mild base treatment to remove ester linkages destroyed FPP's apoptotic activity and yielded homogalacturonan (HG) oligosaccharides. The treatment of FPP with pectinmethylesterase to remove galacturonosyl carboxymethylesters and/or with endopolygalacturonase to cleave nonmethylesterified HG caused no major reduction in apoptotic activity, implicating the requirement for a base-sensitive linkage other than the carboxymethylester. Heat treatment of CP (HTCP) led to the induction of significant levels of apoptosis comparable to FPP, suggesting a means for generating apoptotic pectic structures. These results indicate that specific structural elements within pectin are responsible for the apoptotic activity, and that this structure can be generated, or enriched for, by heat treatment of CP. These findings provide the foundation for mechanistic studies of pectin apoptotic activity and a basis for the development of pectin-based pharmaceuticals, nutraceuticals, or recommended diet changes aimed at combating prostate cancer occurrence and progression.