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Supplementary Table 2

Supplementary figure S4

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5′ untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC.Significance:These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

Supplementary Table 4

Supplementary Table 1

Supplementary Table 3

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5′ untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC.Significance:These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

The oncoprotein and Parkinson's disease-associated enzyme DJ-1/PARK7 has emerged as a promiscuous deglycase that can remove methylglyoxal-induced glycation adducts from both proteins and nucleotides. However, dissecting its structural and enzymatic functions remains a challenge due to the lack of potent, specific, and pharmacokinetically stable inhibitors targeting its catalytic site (including Cys106). To evaluate potential drug-like leads against DJ-1, we leveraged its deglycase activity in an enzyme-coupled, fluorescence lactate-detection assay based on the recent understanding of its deglycation mechanism. In addition, we developed assays to directly evaluate DJ-1's esterase activity using both colorimetric and fluorescent substrates. The resulting optimized assay was used to evaluate a library of potential reversible and irreversible DJ-1 inhibitors. The deglycase activity-oriented screening strategy described herein establishes a new platform for the discovery of potential anti-cancer drugs.

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.

Novel small molecules were synthesized and evaluated as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of inflammatory and autoimmune diseases. A hit compound, 1, was discovered by high-throughput screening of our compound library. The structure-activity relationship (SAR) study of compound 1 showed that the introduction of a chlorine group at the 3-position of 4-cyanophenyl moiety increased the potency and a 3-methylpentane-1,5-diamide linker is favorable for the activity. The carbazole moiety of 1 was also optimized; a quinazolinedione derivative 18i suppressed the increase of IL-17A mRNA level in the lymph node of a rat model of experimental autoimmune encephalomyelitis (EAE) upon oral administration. These results indicate that the novel quinazolinedione derivatives have great potential as orally available small-molecule RORγt inverse agonists for the treatment of Th17-driven autoimmune diseases. A U-shaped bioactive conformation of this chemotype with RORγt protein was also observed.

A series of tetrahydroisoquinoline derivatives were designed, synthesized, and evaluated for their potential as novel orally efficacious retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists for the treatment of Th17-driven autoimmune diseases. We carried out cyclization of the phenylglycinamide core by structure-based drug design and successfully identified a tetrahydroisoquinoline carboxylic acid derivative 14 with good biochemical binding and cellular reporter activity. Interestingly, the combination of a carboxylic acid tether and a central fused bicyclic ring was crucial for optimizing PK properties, and the compound 14 showed significantly improved PK profile. Successive optimization of the carboxylate tether led to the discovery of compound 15 with increased inverse agonistic activity and an excellent PK profile. Oral treatment of mice with compound 15 robustly and dose-dependently inhibited IL-17A production in an IL23-induced gene expression assay.

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein’s unusually large size, four distinct nucleotide-binding sites, and the existence of closely-related isoforms with different functions, pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterizedS. cerevisiaedynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with single molecule experiments and mutagenesis studies, indicate that the compounds likely inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein’s main catalytic site in the AAA1 domain. A double Walker B mutant in AAA3 and AAA4 is an inactive enzyme, suggesting that inhibiting these regulatory sites can have a similar effect to inhibiting AAA1. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein’s AAA domains.

Retinoic-acid-related orphan receptor γt (RORγt) inverse agonists could be used for the treatment of autoimmune diseases. Previously, we reported a novel quinazolinedione 1 a with a flexible linear linker as a novel RORγt inverse agonist. A U-shaped conformation in the complex structure of 1 a with RORγt protein was confirmed. Further improvement of the pharmacokinetic (PK) profiles was required because of the low drug exposure in mice upon oral administration (mouse AUC of 1 a: 27 ng ⋅ h ⋅ mL-1 at 1 mg ⋅ kg-1 , p.o.). To improve the PK profiles, conformationally constrained U-shaped scaffolds were investigated. As a result, morpholine analogues with improved PK profiles and high potency were successfully identified. The substituent at the N1 position of the quinazoline moiety was also modified, leading to an enhancement of reporter activity. Consequently, compound 43 (N2 -(3-chloro-4-cyanophenyl)-N4 -(3-(cyclopropylmethyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydroquinazolin-6-yl)morpholine-2,4-dicarboxamide) exhibited improved drug exposure (mouse AUC: 1289 ng ⋅ h ⋅ mL-1 at 1 mg ⋅ kg-1 , p.o.). In addition, suppression of IL-17A gene expression by IL-23 stimulation in a mouse pharmacodynamics model was observed for 43. The conformation of 43 with RORγt protein was also confirmed as U-shape by X-ray co-crystal structure analysis. The key interaction that boosts potency is also discussed.

[Image: see text] The integrin αVβ3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVβ3-mediated cell adhesion to αVβ3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVβ3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC(50), which correlates with cilengitide’s enhancement of tumor growth in vivo.

The aspartic proteinase renin is an attractive target for the treatment of hypertension and cardiovascular/renal disease such as chronic kidney disease and heart failure. We introduced an S1′ site binder into the lead compound 1 guided by structure-based drug design (SBDD), and further optimization of physicochemical properties led to the discovery of benzimidazole derivative 10 (1-(4-methoxybutyl)-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-yl)carbonylpiperidin-3-yl]-1H-benzimidazole-2-carboxamide hydrochloride, TAK-272) as a highly potent and orally active renin inhibitor. Compound 10 demonstrated good oral bioavailability (BA) and long-lasting efficacy in rats. Compound 10 is currently in clinical trials.

A series of tetrahydronaphthyridine derivatives as novel RORγt inverse agonists were designed and synthesized. We reduced the lipophilicity of tetrahydroisoquinoline compound 1 by replacement of the trimethylsilyl group and SBDD-guided scaffold exchange, which successfully afforded compound 7 with a lower log D value and tolerable in vitro activity. Consideration of LLE values in the subsequent optimization of the carboxylate tether led to the discovery of [ cis-3-({(5 R)-5-[(7-fluoro-1,1-dimethyl-2,3-dihydro-1 H-inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridin-6(5 H)-yl}carbonyl)cyclobutyl]acetic acid, TAK-828F (10), which showed potent RORγt inverse agonistic activity, excellent selectivity against other ROR isoforms and nuclear receptors, and a good pharmacokinetic profile. In animal studies, oral administration of compound 10 exhibited robust and dose-dependent inhibition of IL-17A cytokine expression in a mouse IL23-induced gene expression assay. Furthermore, development of clinical symptoms in a mouse experimental autoimmune encephalomyelitis model was significantly reduced. Compound 10 was selected as a clinical compound for the treatment of Th17-driven autoimmune diseases.

A series of novel phenylglycinamides as retinoic acid receptor-related orphan receptor-gamma t (RORγt) inverse agonists were discovered through optimization of a high-throughput screen hit 1. (R)-N-(2-((3,5-Difluoro-4-(trimethylsilyl)phenyl) amino)-1-(4-methoxyphenyl)-2-oxoethyl)-3-hydroxy-N-methylisoxazole-5-carboxamide (22) was identified as one of the best of these compounds. It displayed higher subtype selectivity and specificity over other nuclear receptors and demonstrated in vivo potency to suppress the transcriptional activity of RORγt in a mouse PD (pharmacodynamic) model upon oral administration.

Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that transport cellular cargos toward microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has in part been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally constrained isosteres. These studies identified dynapyrazoles, inhibitors more potent than ciliobrevins. At single-digit micromolar concentrations dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Further, we find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles strongly block only microtubule-stimulated activity. Together, our studies suggest that chemical-structure-based analyses can lead to inhibitors with improved properties and distinct modes of inhibition. DOI: http://dx.doi.org/10.7554/eLife.25174.001

To elucidate the structural requirements for the endotoxic and antagonistic activities of lipid A derivatives, we have focused on the effects of the acyl moieties and acidic groups at the 1- and 4′...

New lipid A analogs having acidic groups β-glycosidically linked at the 1-position were synthesized in order to investigate the structural requirement for immunostimulating and endotoxic activity of lipid A. The β-(phosphonoxy)ethyl (PE) and carboxymethyl (CM) analogs of Escherichia coli type having six acyl groups and those of the biosynthetic precursor type having four acyl groups were synthesized via a divergent synthetic route. The E. coli type β-(phosphonoxy)ethyl analog, which was previously reported to be not endotoxic, showed strong immunostimulating activity comparable to the natural-type α-analog. The acidic functional groups are concluded to be essential but their strict spatial arrangement is not required for expression of the biological activity.

A new method is described for affinity purification of synthetic compounds based on molecular recognition between bis(2,6-diaminopyridine)amide of isophthalic acid and a barbituric acid derivative. The desired compounds possessing the barbituric acid derivative as a tag were readily isolated from the reaction mix- ture by the following procedure. After each reaction cycle, the reac- tion mixture was applied to the polystyrene column possessing bis(2,6-diaminopyridine)amide of isophthalic acid as an artificial receptor. The compound possessing the barbituric acid tag was se- lectively adsorbed on the column, whereas other impurities without the tag such as excess reagents and byproducts were washed off. Subsequent desorption with CH2Cl2-MeOH (1:1) afforded the de- sired compound with high purity. This new strategy was applied to the synthesis of a heterocycle, peptides, and oligosaccharides.

Both endotoxic and antagonistic [ 3 H]-labeled 2-(phosphonooxy)ethyl (PE) analogs of lipid A were synthesized with high purity and high specific radioactivity. Lipid A-binding proteins were detected by using the endotoxic analog of hexaacyl Escherichia coli -type designated [ 3 H] PE-506. The plasma membrane fractions from peritoneal macrophages derived from LPS-responder C3H/HeN mice and LPS-hyporesponder C3H/HeJ mice were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then incubated with the [ 3 H] PE-506. Several [ 3 H] PE506 binding proteins were detected in both C3H/HeN and C3H/HeJ macrophages. Unlabeled hexaacyl lipid A inhibited the interaction between [ 3 H] PE-506 and these proteins. The result suggests that there exist multiple binding sites for lipid A on macrophages. LPS-induced change in the profile of the cell surface lipid A binding proteins was observed in C3H/HeN macrophages, but not in C3H/HeJ macrophages, by preincubation of macrophages with LPS.

New efficient synthesis of lipid A, an immunostimulat- ing glycoconjugate of bacteria, was achieved for the construction of lipid A library by using synthesis based on affinity separation (SAS), where the compounds possessing a barbituric acid (BA)-tag are selectively and rapidly purified by interaction with an artificial receptor for BA. Glycosylation of a glycosyl acceptor possessing the BA-tag with a 4'-phosphorylated N-Troc glucosaminyl trichlo- roacetimidate gave the disaccharide 4'-phosphate, which was puri- fied by the affinity separation. Successive removal of protective groups and introduction of acyl groups were then effected and the synthetic intermediate at each step was purified by the affinity sep- aration. Cleavage of the tag and subsequent deprotection afforded Escherichia coli lipid A. SAS enabled the rapid preparation of lipid A, therefore, proved to be a promising method for synthesis of other complex glycoconjugates.

In order to investigate precise structural requirements for expression of the biological activity of lipid A, new structural analogs were synthesized via improved, highly efficient preparative routes. The number and chain length of the fatty acyl groups proved to have crucial influence on the biological activity. For the study of conformation relevant to activity of lipid A by means of NMR spectrometry, lipid A analogs [13C]-labeled at the 6-position were then synthesized starting from 6-[13C]-glucose. Precise analysis of J CH and NOE data disclosed a particular molecular conformation and characteristic supramolecular assembly in an aqueous SDS micelle solution of the tetraacyl biosynthetic precursor molecule. The observed mode of assembly may reflect the actual behavior of lipid A molecules in the cell surface outer membrane of living bacteria. A biologically inactive, artificial, tetra-acyl analog having shorter acyl chains exhibits, by contrast, distinct conformations with no sign of supramolecule formation. The acyl moieties in lipid A are thus found to play an important role in regulating the overall conformation of the hydrophilic backbone.

Novel lipid A analogues which possess four ( R )-3-hydroxyacyl moieties of shorter chain length were synthesized via a new divergent synthetic route in order to clarify the effect of the chain length of acyl groups to the biological activity: a disaccharide 4′-phosphate was first constructed as a common synthetic intermediate and all acyl moieties were then introduced step by step to the respective positions. The hydroxy group in acyl moieties was protected with a benzyl group by novel 1-pot reductive alkylation using benzaldehyde, TMS 2 O, TMSOTf, and Et 3 SiH. Both the glycosyl donor and acceptor were synthesized by using the new method recently reported by ourselves for the regioselective reductive opening of 4,6- O -benzylidene glucosamine derivatives with BH 3 ·Me 2 NH and BF 3 ·OEt 2 . In this reaction, a 3- O -allyloxycarbonylated 4,6- O -benzylidene compound in CH 3 CN afforded the 6- O -benzylated product selectively, which was then converted to a glycosyl trichloroacetimidate used as the donor. The 4- O -benzylated acceptor was synthesized by the same reductive opening of a 3- O -p-methoxybenzylated compound in CH 2 Cl 2 . A disaccharide 4′-phosphate was synthesized by coupling of the imidate donor and the acceptor using TMSOTf as a catalyst. ( R )-3-Benzyloxyacyl groups were then introduced to the 3,3′, 2 and 2′ positions followed by 1- O -phosphorylation and subsequent deprotection by Pd H 2 afforded the desired lipid A analogues. The present divergent route opens an efficient way toward the synthesis of lipid A libraries. Biological tests (inhibition of IL-6 induction) clearly showed the critical importance of the chain length of the acyl moieties in lipid A to the activity.

Novel lipid A analogues which possess four ( R )-3-hydroxyacyl moieties of shorter chain length were synthesized via a new divergent synthetic route in order to clarify the effect of the chain length of acyl groups to the biological activity: a disaccharide 4′-phosphate was first constructed as a common synthetic intermediate and all acyl moieties were then introduced step by step to the respective positions. The hydroxy group in acyl moieties was protected with a benzyl group by novel 1-pot reductive alkylation using benzaldehyde, TMS 2 O, TMSOTf, and Et 3 SiH. Both the glycosyl donor and acceptor were synthesized by using the new method recently reported by ourselves for the regioselective reductive opening of 4,6- O -benzylidene glucosamine derivatives with BH 3 ·Me 2 NH and BF 3 ·OEt 2 . In this reaction, a 3- O -allyloxycarbonylated 4,6- O -benzylidene compound in CH 3 CN afforded the 6- O -benzylated product selectively, which was then converted to a glycosyl trichloroacetimidate used as the donor. The 4- O -benzylated acceptor was synthesized by the same reductive opening of a 3- O -p-methoxybenzylated compound in CH 2 Cl 2 . A disaccharide 4′-phosphate was synthesized by coupling of the imidate donor and the acceptor using TMSOTf as a catalyst. ( R )-3-Benzyloxyacyl groups were then introduced to the 3,3′, 2 and 2′ positions followed by 1- O -phosphorylation and subsequent deprotection by Pd H 2 afforded the desired lipid A analogues. The present divergent route opens an efficient way toward the synthesis of lipid A libraries. Biological tests (inhibition of IL-6 induction) clearly showed the critical importance of the chain length of the acyl moieties in lipid A to the activity.

The propargyloxycarbonyl group readily introduced to both amino and hydroxy groups by using propargyl chloroformate is stable to neat TFA but is readily cleaved at ambient temperature by treatment with Co 2 (CO) 8 and TFA in CH 2 Cl 2 via formation of an alkyne-Co complex. The propargyl ester similarly serves as a good protecting group for carboxy functions.

In order to investigate precise structural requirements for expression of the biological activity of lipid A, new structural analogs were synthesized via improved, highly efficient preparative rout...

New efficient synthesis of lipid A, an immunostimulat- ing glycoconjugate of bacteria, was achieved for the construction of lipid A library by using synthesis based on affinity separation (SAS), where the compounds possessing a barbituric acid (BA)-tag are selectively and rapidly purified by interaction with an artificial receptor for BA. Glycosylation of a glycosyl acceptor possessing the BA-tag with a 4'-phosphorylated N-Troc glucosaminyl trichlo- roacetimidate gave the disaccharide 4'-phosphate, which was puri- fied by the affinity separation. Successive removal of protective groups and introduction of acyl groups were then effected and the synthetic intermediate at each step was purified by the affinity sep- aration. Cleavage of the tag and subsequent deprotection afforded Escherichia coli lipid A. SAS enabled the rapid preparation of lipid A, therefore, proved to be a promising method for synthesis of other complex glycoconjugates.

To elucidate the structural requirements for the endotoxic and antagonistic activities of lipid A derivatives, we have focused on the effects of the acyl moieties and acidic groups at the 1- and 4′...

The detailed conformational analysis of a single molecule of the tetraacyl biosynthetic precursor-type lipid A and its characteristic supramolecular assembly in aqueous SDS-micelles are described. Regular molecular arrangements were observed by detailed analysis of the NMR spectra of synthetically pure specimens, including regiospecifically 13C-labeled ones. NMR analysis of a biologically inactive precursor-type analogue with four shorter acyl chains demonstrated its conformational flexibility, indicating the importance of hydrophobic interactions for maintaining the conformation of such molecules.

The propargyloxycarbonyl group readily introduced to both amino and hydroxy groups by using propargyl chloroformate is stable to neat TFA but is readily cleaved at ambient temperature by treatment with Co 2 (CO) 8 and TFA in CH 2 Cl 2 via formation of an alkyne-Co complex. The propargyl ester similarly serves as a good protecting group for carboxy functions.