Discovery and optimization of substituted oxalamides as novel heme-displacing IDO1 inhibitors.

Since the advent of antibody checkpoint inhibitors as highly efficient drugs for cancer treatment, the development of immunomodulating small molecules in oncology has gained great attention. Drug candidates targeting IDO1, a key enzyme in tryptophan metabolism, are currently under clinical investigation in combination with PD-1/PD-L1 agents as well as with other established anti-tumor therapeutics. A ligand based design approach from hydroxyamidine 4 that aimed at heme-binding IDO1 inhibitors resulted in new compounds with moderate IDO1 potency. A hybrid structure design that made use of the linrodostat structure (2) led to oxalamide derived, heme-displacing IDO1 inhibitors with high cell-based IDO1 potency and a favorable ADME/PK profile.

Discovery of highly potent heme-displacing IDO1 inhibitors based on a spirofused bicyclic scaffold.

Through structural modification of an oxalamide derived chemotype, a novel class of highly potent, orally bioavailable IDO1-specific inhibitors was identified. Representative compound 18 inhibited human IDO1 with IC50 values of 3.9 nM and 52 nM in a cellular and human whole blood assay, respectively. In vitro assessment of the ADME properties of 18 demonstrated very high metabolic stability. Pharmacokinetic profiling in mice showed a significantly reduced clearance compared to the oxalamides. In a mouse pharmacodynamic model 18 nearly completely suppressed lipopolysaccharide-induced kynurenine production. Hepatocyte data of 18 suggest the human clearance to be in a similar range to linrodostat (1).

Optimization and biological evaluation of thiazole-bis-amide inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with small molecule therapeutics. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We recently described the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole scaffold. Herein we describe the successful optimization of this class by incorporation of an additional amide moiety at the 4-position of the thiazole core. In several optimization cycles, we have reduced human PXR activation, improved solubility, and increased potency while maintaining nuclear receptor selectivity. X-ray crystallographic analysis of compound 1g bound in the sterol binding site of the ligand binding domain of RORγt was largely consistent with an earlier structure, guiding further insight into the molecular mechanism for RORγt inhibition with this series. Compound 1g is orally bioavailable, potent in a human whole blood assay and proved to be efficacious in an ex-vivo IL-17A assay, and was selected for preclinical evaluation.

Discovery and optimization of new oxadiazole substituted thiazole RORγt inverse agonists through a bioisosteric amide replacement approach.

Starting from previously identified thiazole-2-carboxamides exemplified by compound 1/6, two new series of RORγt inverse agonists with significantly improved aqueous solubility, ADME parameters and oral PK properties were discovered. These scaffolds were identified from a bioisosteric amide replacement approach. Amongst the variety of heterocycles explored, a 1,3,4-oxadiazole led to compounds with the best overall profile for SAR development and in vivo exploration. In an ex vivo mouse PD model, concentration dependent efficacy was demonstrated and compounds 3/5 and 6/3 were profiled in a 5-day rat tolerability study.

3-Substituted Quinolines as RORγt Inverse Agonists.

We have previously reported the syntheses of a series of 3,6-disubstituted quinolines as modulators of the retinoic acid receptor-related orphan receptor gamma t (RORγt). These molecules are potent binders but are high molecular weight and they exhibited poor solubility at pH 2 and pH 7. This manuscript details our efforts at improving physical chemical properties for this series of compounds by increasing the diversity at the 3-position (i.e. introducing heteroatoms and lowering the molecular weight). These efforts have led to molecules which are potent binders with improved solubility.

Photoactivated Colibactin Probes Induce Cellular DNA Damage.

Colibactin is a small molecule produced by certain bacterial species of the human microbiota that harbour the pks genomic island. Pks+ bacteria induce a genotoxic phenotype in eukaryotic cells and have been linked with colorectal cancer progression. Colibactin is produced in a benign, prodrug form which, prior to export, is enzymatically matured by the producing bacteria to its active form. Although the complete structure of colibactin has not been determined, key structural features have been described including an electrophilic cyclopropane motif, which is believed to alkylate DNA. To investigate the influence of the putative "warhead" and the prodrug strategy on genotoxicity, a series of photolabile colibactin probes were prepared that upon irradiation induced a pks+ like phenotype in HeLa cells. Furthermore, results from DNA cross-linking and imaging studies of clickable analogues enforce the hypothesis that colibactin effects its genotoxicity by directly targeting DNA.

Identification and biological evaluation of thiazole-based inverse agonists of RORγt.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma t (RORγt) is a transcription factor that drives Th17 cell differentiation and IL-17 production in both innate and adaptive immune cells. The IL-23/IL-17 pathway is implicated in major autoimmune and inflammatory diseases. RORγt lies at the core of this pathway and represents an attractive opportunity for intervention with a small molecule. Despite diverse chemical series having been reported, combining high potency and nuclear receptor selectivity with good physicochemical properties remains a challenging endeavor in the field of RORγt drug discovery. We describe the discovery and evaluation of a new class of potent and selective RORγt inverse agonists based on a thiazole core. Acid analog 1j demonstrated oral bioavailability in rats and was potent in a human whole blood assay, suggesting potential utility in treating autoimmune and inflammatory diseases such as psoriasis. X-ray crystallographic data helped to elucidate the molecular mechanism for RORγt inhibition with this series.

The α-hydroxyketone LAI-1 regulates motility, Lqs-dependent phosphorylation signalling and gene expression of Legionella pneumophila.

The causative agent of Legionnaires' disease, Legionella pneumophila, employs the autoinducer compound LAI-1 (3-hydroxypentadecane-4-one) for cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, comprising the autoinducer synthase LqsA, the sensor kinases LqsS and LqsT, as well as the response regulator LqsR. Lqs-regulated processes include pathogen-host interactions, production of extracellular filaments and natural competence for DNA uptake. Here we show that synthetic LAI-1 promotes the motility of L. pneumophila by signalling through LqsS/LqsT and LqsR. Upon addition of LAI-1, autophosphorylation of LqsS/LqsT by [γ-(32) P]-ATP was inhibited in a dose-dependent manner. In contrast, the Vibrio cholerae autoinducer CAI-1 (3-hydroxytridecane-4-one) promoted the phosphorylation of LqsS (but not LqsT). LAI-1 did neither affect the stability of phospho-LqsS or phospho-LqsT, nor the dephosphorylation by LqsR. Transcriptome analysis of L. pneumophila treated with LAI-1 revealed that the compound positively regulates a number of genes, including the non-coding RNAs rsmY and rsmZ, and negatively regulates the RNA-binding global regulator crsA. Accordingly, LAI-1 controls the switch from the replicative to the transmissive growth phase of L. pneumophila. In summary, the findings indicate that LAI-1 regulates motility and the biphasic life style of L. pneumophila through LqsS- and LqsT-dependent phosphorylation signalling.

Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway.

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9.

Covalent Protein Labeling by Enzymatic Phosphocholination.

We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.

Exploring adenylylation and phosphocholination as post-translational modifications.

Editing the translations: Adenylylation and phosphocholination have recently been found as important post-translational modifications used by pathogenic bacteria during the infection process. This review discusses the combined use of chemical handles and specific antibodies for the identification of previously unknown substrates of these post-translational modifications in infected host cells.

Amino acid building blocks for Fmoc solid-phase synthesis of peptides phosphocholinated at serine, threonine, and tyrosine.

Phosphocholination of eukaryotic host cell proteins has recently been identified as a novel post-translational modification important for bacterial pathogenesis. Here, we describe the first straightforward synthetic strategy for peptides containing phosphocholinated serine, threonine, or tyrosine residues using preformed functional amino acid building blocks, fully compatible with standard Fmoc solid-phase peptide synthesis.

Mechanism of the tissue-specific action of the selective androgen receptor modulator S-101479.

Selective androgen receptor modulators (SARMs) comprise a new class of molecules that induce anabolic effects with fewer side effects than those of other anabolic agents. We previously reported that the novel SARM S-101479 had a tissue-selective bone anabolic effect with diminished side effects in female animals. However, the mechanism of its tissue selectivity is not well known. In this report, we show that S-101479 increased alkaline phosphatase activity and androgen receptor (AR) transcriptional activity in osteoblastic cell lines in the same manner as the natural androgen ligand dihydrotestosterone (DHT); conversely, stimulation of AR dimerization was very low compared with that of DHT (34.4%). S-101479 increased bone mineral content in ovariectomized rats without promoting endometrial proliferation. Yeast two-hybrid interaction assays revealed that DHT promoted recruitment of numerous cofactors to AR such as TIF2, SRC1, ß-catenin, NCoA3, gelsolin and PROX1 in a dose-dependent manner. SARMs induced recruitment of fewer cofactors than DHT; in particular, S-101479 failed to induce recruitment of canonical p160 coactivators such as SRC1, TIF2 and notably NCoA3 but only stimulated binding of AR to gelsolin and PROX1. The results suggest that a full capability of the AR to dimerize and to effectively and unselectively recruit all canonical cofactors is not a prerequisite for transcriptional activity in osteoblastic cells and resulting anabolic effects in bone tissues. Instead, few relevant cofactors might be sufficient to promote AR activity in these tissues.

Preclinical and clinical characterization of the RORγt inhibitor JNJ-61803534.

The nuclear receptor retinoid-related orphan receptor gamma t (RORγt) plays a critical role in driving Th17 cell differentiation and expansion, as well as IL-17 production in innate and adaptive immune cells. The IL-23/IL-17 axis is implicated in several autoimmune and inflammatory diseases, and biologics targeting IL-23 and IL-17 have shown significant clinical efficacy in treating psoriasis and psoriatic arthritis. JNJ-61803534 is a potent RORγt inverse agonist, selectively inhibiting RORγt-driven transcription versus closely-related family members, RORα and RORß. JNJ-61803534 inhibited IL-17A production in human CD4+ T cells under Th17 differentiation conditions, but did not inhibit IFNγ production under Th1 differentiation conditions, and had no impact on in vitro differentiation of regulatory T cells (Treg), nor on the suppressive activity of natural Tregs. In the mouse collagen-induced arthritis model, JNJ-61803534 dose-dependently attenuated inflammation, achieving ~ 90% maximum inhibition of clinical score. JNJ-61803534 significantly inhibited disease score in the imiquimod-induced mouse skin inflammation model, and dose-dependently inhibited the expression of RORγt-regulated genes, including IL-17A, IL-17F, IL-22 and IL-23R. Preclinical 1-month toxicity studies in rats and dogs identified doses that were well tolerated supporting progression into first-in-human studies. An oral formulation of JNJ-61803534 was studied in a phase 1 randomized double-blind study in healthy human volunteers to assess safety, pharmacokinetics, and pharmacodynamics. The compound was well tolerated in single ascending doses (SAD) up to 200 mg, and exhibited dose-dependent increases in exposure upon oral dosing, with a plasma half-life of 164 to 170 h. In addition, dose-dependent inhibition of ex vivo stimulated IL-17A production in whole blood was observed, demonstrating in vivo target engagement. In conclusion, JNJ-61803534 is a potent and selective RORγt inhibitor that exhibited acceptable preclinical safety and efficacy, as well as an acceptable safety profile in a healthy volunteer SAD study, with clear evidence of a pharmacodynamic effect in humans.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor alpha.

In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERalpha) in an agonist-dependent manner. The interaction is specific for ERalpha as ERbeta and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERalpha was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERalpha is sufficient for interaction with RAP80. RAP80 overexpression reduces ERalpha polyubiquitination, increases the level of ERalpha protein, and enhances ERalpha-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERalpha protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERalpha, but eliminates the effects of RAP80 on ERalpha polyubiquitination, the level of ERalpha protein, and ERalpha-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERalpha as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERalpha activity.

Neurotrophic estrogens: essential profile and endpoints for drug discovery.

Criteria for the early recognition of selective neurotrophic action are crucial for the discovery of estrogens for supplementation therapy. The comparative characterization of 'tool' compounds in different paradigms demonstrates that estrogen-mediated CNS effects are discernible before the manifestation of changes in primary target organs. Agonist activity at, and recruitment of the coactivator SRC-1 by, the estrogen receptor alpha accurately reflect peripheral, but not neurotrophic, efficacy. Interaction with, and SRC-1 recruitment at, the estrogen receptor beta appears to be an essential prerequisite for pronounced CNS effects. Monitoring of the hypothalamo-pituitary-adrenal axis activity and the differential organ-specific induction of estrogen-responsive proteins are helpful for early delineation of CNS efficacy. Behavioral and antioxidant efficacy are useful confirmatory readouts, with limited roles in lead selection. Finally, an algorithm for the identification of estrogens with a neurotrophic profile can be generated by assigning 'performance grades' in a multifarious test array.

Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas).

An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H(2)O(2) at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 10(3)-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72).

Regulation of IL-22BP in psoriasis.

IL-22 is a potent pro-inflammatory cytokine upregulated in psoriasis and in other inflammatory diseases. The function of IL-22 is regulated by the soluble scavenging receptor, IL-22 binding protein (IL-22BP or IL-22RA2). However, the role and regulation of IL-22BP itself in the pathogenesis of inflammatory disease remain unclear. We used the TLR7 agonist Imiquimod (IMQ) to induce a psoriasis-like skin disease in mice and found a strong downregulation of IL-22BP in the affected skin as well as in the lymph nodes of animals treated with IMQ. We also analysed psoriatic skin of patients and compared this to skin of healthy donors. Interestingly, IL-22BP expression was similarly downregulated in skin biopsies of psoriasis patients compared to the skin of healthy donors. Since IL-22BP is expressed foremost in dendritic cells, we characterized its expression in monocyte-derived dendritic cells (MoDC) during maturation. In this way, we found Prostaglandin E2 (PGE2) to be a potent suppressor of IL-22BP expression in vitro. We conclude that regulation of IL-22BP by inflammatory mediators is an important step for the progression of inflammation in the skin and possibly also in other autoimmune diseases.

Panning for SNuRMs: using cofactor profiling for the rational discovery of selective nuclear receptor modulators.

Drugs that target nuclear receptors are clinically, as well as commercially, successful. Their widespread use, however, is limited by an inherent propensity of nuclear receptors to trigger beneficial, as well as adverse, pharmacological effects upon drug activation. Hence, selective drugs that display reduced adverse effects, such as the selective estrogen receptor modulator (SERM) Raloxifene, have been developed by guidance through classical cell culture assays and animal trials. Full agonist and selective modulator nuclear receptor drugs, in general, differ by their ability to recruit certain cofactors to the receptor protein. Hence, systematic cofactor profiling is advancing into an approach for the rationally guided identification of selective NR modulators (SNuRMs) with improved therapeutic ratio.