Design and synthesis of tetrahydropyridopyrimidine based Toll-Like Receptor (TLR) 7/8 dual agonists

In a continuing effort to discover novel TLR agonists, herein we report on the discovery and structure–activity relationship of novel tetrahydropyridopyrimidine TLR 7/8 agonists. Optimization of this series towards dual agonist activity and a high clearance profile resulted in the identification of compound 52a1. Evaluation in vivo revealed an interferon stimulated response (ISG) in mice with limited systemic exposure and demonstrated the potential in antiviral treatment or as a vaccine adjuvant.     

Discovery of selective 2,4-diaminoquinazoline toll-like receptor 7 (TLR 7) agonists

The discovery of a novel series of highly potent quinazoline TLR 7/8 agonists is described. The synthesis and structure–activity relationship is presented. Structural requirements and optimization of this series toward TLR 7 selectivity afforded the potent agonist 48. Pharmacokinetic and pharmacodynamic studies highlighted 48 as an orally available endogenous interferon (IFN-α) inducer in mice.     

Identification and optimization of indolo[2,3-c]quinoline inhibitors of IRAK4

IRAK4 is responsible for initiating signaling from Toll-like receptors (TLRs) and members of the IL-1/18 receptor family. Kinase-inactive knock-ins and targeted deletions of IRAK4 in mice cause reductions in TLR induced pro-inflammatory cytokines and these mice are resistant to various models of arthritis. Herein we report the identification and optimization of a series of potent IRAK4 inhibitors. Representative examples from this series showed excellent selectivity over a panel of kinases, including the kinases known to play a role in TLR-mediated signaling. The compounds exhibited low nM potency in LPS- and R848-induced cytokine assays indicating that they are blocking the TLR signaling pathway. A key compound (26) from this series was profiled in more detail and found to have an excellent pharmaceutical profile as measured by predictive assays such as microsomal stability, TPSA, solubility, and c log P. However, this compound was found to afford poor exposure in mouse upon IP or IV administration. We found that removal of the ionizable solubilizing group (32) led to increased exposure, presumably due to increased permeability. Compounds 26 and 32, when dosed to plasma levels corresponding to ex vivo whole blood potency, were shown to inhibit LPS-induced TNFα in an in vivo murine model. To our knowledge, this is the first published in vivo demonstration that inhibition of the IRAK4 pathway by a small molecule can recapitulate the phenotype of IRAK4 knockout mice.     

Identification and immunological evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4

Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2 agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective agonists, a novel series of Pam₃CSK₄ derivatives were designed based on the crystal structure of hTLR2-hTLR1-Pam₃CSK₄ complex, synthesized and evaluated for their immune-stimulatory activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2 agonistic activity comparable to Pam₃CSK₄, which included: elevated IL-6 expression level (EC₅₀ = 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and 35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.     

Surrogating and redirection of pyrazolo[1,5-a]pyrimidin-7(4H)-one core,a novel class of potent and selective DPP-4 inhibitors

The initial focus on characterizing novel pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as DPP-4 inhibitors, led to a potent and selective inhibitor compound b2. This ligand exhibits potent in vitro DPP-4 inhibitory activity (IC₅₀: 80?nM), while maintaining other key cellular parameters such as high selectivity, low cytotoxicity and good cell viability. Subsequent optimization of b2 based on docking analysis and structure-based drug design knowledge resulted in d1. Compound d1 has nearly 2-fold increase of inhibitory activity (IC₅₀: 49?nM) and over 1000-fold selectivity against DPP-8 and DPP-9. Further in vivo IPGTT assays showed that compound b2 effectively reduce glucose excursion by 34% at the dose of 10?mg/kg in diabetic mice. Herein we report the optimization and design of a potent and highly selective series of pyrazolo[1,5-a]pyrimidin-7(4H)-one DPP-4 inhibitors.     

Cardiac RNA Induces Inflammatory Responses in Cardiomyocytes and Immune Cells via Toll-like Receptor 7 Signaling

We have recently reported that extracellular RNA (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes and immune cells and contributes to myocardial ischemia/reperfusion injury. However, the signaling mechanism by which exRNA exhibits its pro-inflammatory effect is unknown. Here we hypothesize that exRNA directly induces inflammation through specific Toll-like receptors (TLRs). To test the hypothesis, we treated rat neonatal cardiomyocytes, mouse bone marrow-derived macrophages (BMDM), or mouse neutrophils with RNA (2.5–10 μg/ml) isolated from rat cardiomyocytes or the hearts from mouse, rat, and human. We found that cellular RNA induced production of several cytokines such as macrophage inflammatory protein-2 (MIP-2), ILs, TNFα, and the effect was completely diminished by RNase, but not DNase. The RNA-induced cytokine production was partially inhibited in cells treated with TLR7 antagonist or genetically deficient in TLR7. Deletion of myeloid differentiation primary response protein 88 (MyD88), a downstream adapter of TLRs including TLR7, abolished the RNA-induced MIP-2 production. Surprisingly, genetic deletion of TLR3 had no impact on the RNA-induced MIP-2 response. Importantly, extracellular RNA released from damaged cardiomyocytes also induced cytokine production. Finally, mice treated with 50 μg of RNA intraperitoneal injection exhibited acute peritonitis as evidenced by marked neutrophil and monocyte migration into the peritoneal space. Together, these data demonstrate that exRNA of cardiac origin exhibits a potent pro-inflammatory property in vitro and in vivo and that exRNA induces cytokine production through TLR7-MyD88 signaling.     

The ex vivo toll-like receptor 7 tolerance induction in donor lymphocytes prevents murine acute graft-versus-host disease

Background aims

Acute graft-versus-host disease (aGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation, mediated by alloreactive donor T cells. Toll-like receptors (TLRs), a family of conserved pattern-recognition receptors (PRRs), represent key players in donors' T-cell activation during aGVHD; however, a regulatory, tolerogenic role for certain TLRs has been recognized in a different context. We investigated whether the ex vivo–induced TLR-2,-4,-7 tolerance in donor cells could prevent alloreactivity in a mismatched transplantation model.

Optimization of 8-oxoadenines with toll-like-receptor 7 and 8 activity

Toll-like receptors 7 and 8 (TLR7/8) agonists are potent immunostimulants that are attracting considerable interest as vaccine adjuvants. We recently reported the synthesis of a new series of 2-O-butyl-8-oxoadenines substituted at the 9-position with various linkers and N-heterocycles, and showed that TLR7/8 selectivity, potency and cytokine induction could be modulated by varying the alkyl linker length and the N-heterocyclic ring. In the present study, we further optimized the oxoadenine scaffold by investigating the effect of different substituents at the 2-position of the oxoadenine on TLR7/8 potency/selectivity, cytokine induction and DC maturation in human PBMCs. The results show that introducing a 1-(S)-methylbutoxy group at the 2-position of the oxoadenine significantly increased potency for TLR7/8 activity, cytokine induction and DC maturation.     

Synthesis and anti-OXPHOS,antitumor activities of DLC modified spinosyn derivatives

A series of DLC (delocalized lipophilic cation) modified spinosyn derivatives were synthesized and evaluated for antitumor efficacies both in vitro and in vivo. Cancer cell based antiproliferative assays indicated that the more lipophilic derivatives had stronger inhibitory effects on the tested cancer cell lines. Compound 7b and 8b exhibited strong anti-OXPHOS and apoptosis inducing ability. Notable antitumor efficacies of 7b (5 mg/kg) and 8b (2.5 mg/kg) were observed in the in vivo tumor xenograft experiments, however, lethal toxicities were observed on higher dosages. Our findings indicated that DLC modification is a viable strategy to enhance the anti-OXPHOS and antitumor efficacies of spinosyn derivatives.     

COX-1/COX-2 inhibition assays and histopathological study of the new designed anti-inflammatory agent with a pyrazolopyrimidine core

Four pyrazolopyrimidine series were prepared with a substitution at position- 4 by Schiff base, triazole, oxadiazole and pyrazole moieties (7a-f, 8a,b, 9a-f, 10a,b and 13a,b), respectively. All the synthesized compounds were evaluated in vitro against COX-2 and in vivo against carrageenan-induced rat paw edema as anti-inflammatory agents. Regarding the anti-inflammatory activity (AI) compounds 7c, 7f, 8a, and 9a showed higher activity with respect to celecoxib. Compounds 9a, 7d, and 7f were closely selective to celecoxib. Also, 7c and 7d were safer than indomethacin and similar to celecoxib as resulted from the histopathological study. In addition, the docking study that showed the binding mode of prominent pyrazolopyrimidine compounds inside the COX-2 receptor. Formation of unexpected pyrazole 13a and 13b was briefly discussed using 2D NMR.     

Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.     

Antimalarial activity of novel 4-aminoquinolines active against drug resistant strains

In the present study we have synthesized a new class of 4-aminoquinolines and evaluated against Plasmodium falciparum in vitro (3D7-sensitive strain & K1-resistant strain) and Plasmodium yoelii in vivo (N-67 strain). Among the series, eleven compounds (5, 6, 7, 8, 9, 11, 12, 13, 14, 15 and 21) showed superior antimalarial activity against K1 strain as compared to CQ. In addition, all these analogues showed 100% suppression of parasitemia on day 4 in the in vivo mouse model against N-67 strain when administered orally. Further, biophysical studies suggest that this series of compounds act on heme polymerization target.     

Design,synthesis and evaluation of novel N-phenylbutanamide derivatives as KCNQ openers for the treatment of epilepsy

KCNQ (Kv7) has emerged as a validated target for the development of novel anti-epileptic drugs. In this paper, a series of novel N-phenylbutanamide derivatives were designed, synthesized and evaluated as KCNQ openers for the treatment of epilepsy. These compounds were evaluated for their KCNQ opening activity in vitro and in vivo. Several compounds were found to be potent KCNQ openers. Compound 1 with favorable in vitro activity was submitted to evaluation in vivo. Results showed that compound 1 owned significant anti-convulsant activity with no adverse effects. It was also found to posses favorable pharmacokinetic profiles in rat. This research may provide novel potent compounds for the discovery of KCNQ openers in treating epilepsy.     

Discovery of a novel selective water-soluble SMAD3 inhibitor as an antitumor agent

Targeting the SMAD3 protein is an attractive therapeutic strategy for treating cancer, as it avoids the potential toxicities due to targeting the TGF-β signaling pathway upstream. Compound SIS3 was the first selective SMAD3 inhibitor developed that had acceptable activity, but its poor water solubility limited its development. Here, a series of SIS3 analogs was created to investigate the structure–activity relationship for inhibiting the activation of SMAD3. On the basis of this SAR, further optimization generated a water-soluble compound, 16d, which was capable of effectively blocking SMAD3 activation in vitro and had similar NK cell-mediated anticancer effects in vivo to its parent SIS3. This study not only provided a preferable lead compound, 16d, for further drug discovery or a potential tool to study SMAD3 biology, but also proved the effectiveness of our strategy for water-solubility driven optimization.     

Role of Toll-Like Receptors 2 and 4 in Pulmonary Inflammation and Injury Induced by Pneumolysin in Mice

Background

Pneumolysin (PLN) is an intracellular toxin of Streptococcus pneumoniae that has been implicated as a major virulence factor in infections caused by this pathogen. Conserved bacterial motifs are recognized by the immune system by pattern recognition receptors among which the family of Toll-like receptors (TLRs) prominently features. The primary objective of the present study was to determine the role of TLR2 and TLR4 in lung inflammation induced by intrapulmonary delivery of PLN.

Methodology/Results

First, we confirmed that purified PLN activates cells via TLR4 (not via TLR2) in vitro, using human embryonic kidney cells transfected with either TLR2 or TLR4. Intranasal administration of PLN induced an inflammatory response in the pulmonary compartment of mice in vivo, as reflected by influx of neutrophils, release of proinflammatory cytokines and chemokines, and a rise in total protein concentrations in bronchoalveolar lavage fluid. These PLN-induced responses were dependent in part, not only on TLR4, but also on TLR2, as indicated by studies using TLR deficient mice.

Conclusion

These data suggest that although purified PLN is recognized by TLR4 in vitro, PLN elicits lung inflammation in vivo by mechanisms that may involve multiple TLRs.     

Synthetic oligoribonucleotides-containing secondary structures act as agonists of Toll-like receptors 7 and 8

Single-stranded RNAs act as ligands of Toll-like receptors (TLRs) 7 and 8 and induce immune responses. In the present study, we have designed and synthesized phosphorothioate oligoribonucleotides (ORNs) with self-complementary sequences that form duplex structures with either 3′- or 5′-overhanging sequences. We studied the new ORNs for their duplex formation, nuclease stability, and ability to induce immune-stimulatory activate through TLR7 and TLR8 in TLR-transfected cell lines, human PBMCs, human pDCs, and in vivo in mice. Thermal melting and gel electrophoresis studies showed that all ORNs formed secondary structures and that the thermal stability of the duplex is depended on the length and GC composition of the duplex. Nuclease stability of ORNs increased with increasing thermal stability of the duplex formed. All ORN showed TLR8 activity in HEK293 cells, and induced cytokine and chemokine production in human PBMC cultures. In addition to TLR8 activity, two ORNs containing a ‘CUGAAUU’ motif in the duplex-forming region induced immune stimulation through TLR7 in HEK293 cells, human PBMC and pDC cultures, and in vivo in mice. These results suggest that secondary structures in ORN provide nuclease stability and lead to stimulation of immune responses through TLR8 as well as TLR7 depending on the presence of specific nucleotide motifs.     

Discovery of 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as potent,selective and orally bioavailable LRRK2 inhibitors

Inhibition of LRRK2 kinase activity with small molecules has emerged as a potential novel therapeutic treatment for Parkinson’s disease. Herein we disclose the discovery of a 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amine series as potent LRRK2 inhibitors identified through a kinase-focused set screening. Optimization of the physicochemical properties and kinase selectivity led to the discovery of compound 7, which exhibited potent in vitro inhibition of LRRK2 kinase activity, good physicochemical properties and kinase selectivity across the kinome. Moreover, compound 7 was able to penetrate into the CNS, and in vivo pharmacology studies revealed significant inhibition of Ser935 phosphorylation in the brain of both rats (30 and 100?mg/kg) and mice (45?mg/kg) following oral administration.     

Molecular characterization of toll-like receptor 2 (TLR2), analysis of its inductive expression and associated down-stream signaling molecules following ligands exposure and bacterial infection in the Indian major carp, rohu (Labeo rohita)

Toll-like receptors (TLRs) are one of the key components of innate immunity. Among various TLR types, TLR2 is involved in recognizing specific microbial structures such as peptidoglycan (PGN), lipoteichoic acid (LTA), zymosan etc., and after binding them it triggers myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathway to induce various cytokines. In this report, TLR2 gene was cloned and characterized in rohu (Labeo rohita), which is highly commercially important fish species in the farming-industry of Indian subcontinent. Full-length rohu TLR2 (rTLR2) cDNA comprised of 2691 bp with a single open reading frame (ORF) of 2379 bp encoding a polypeptide of 792 amino acids (aa) with an estimated molecular mass of 90.74 kDa. Structurally, it comprised of one leucine-rich repeat region (LRR) each at N-terminal (LRR-NT; 44-55 aa) and C-terminal (LRR-CT; 574-590 aa), 21 LRRs in between C and N-terminal, one trans-membrane (TM) domain (595-612 aa), and one TIR domain (645-790 aa). Phylogenetically, rohu TLR2 was closely related to common carp and exhibited significant similarity (93.1%) and identity (88.1%) in their amino acids. During embryogenesis, rTLR2 expression was detected as early as ∼7 h post fertilization indicating its importance in embryonic innate immune defense system in fish. Basal expression analysis of rTLR2 showed its constitutive expression in all the tissues examined, highest was in the spleen and the lowest was in the eye. Inductive expression of TLR2 was observed following zymosan, PGN and LTA exposure and Streptococcus uberis and Edwardsiella tarda infections. Expression of immunoregulatory cytokine interleukin (IL)-8, in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with inductive expression of TLR2. In vitro studies showed that PGN treatment induced TLR2, MyD88 and TRAF6 (TNF receptor associated factor 6) expression, NF-κB (nuclear factor kappa B) activation and IL-8 expression. Blocking NF-κB resulted in down-regulation of PGN mediated IL-8 expression indicating the involvement of NF-κB in IL-8 induction. Together, these findings highlighted the important role of TLR2 in immune surveillance of various organs, and in augmenting innate immunity in fish in response to pathogenic invasion. This study will be helpful in developing preventive measures against infectious diseases in fish.     

Design,synthesis and biological evaluation of novel pyrimidinedione derivatives as DPP-4 inhibitors

A series of novel pyrimidinedione derivatives were designed and evaluated for in vitro dipeptidyl peptidase-4 (DPP-4) inhibitory activity and in vivo anti-hyperglycemic efficacy. Among them, the representative compounds 11, 15 and 16 showed excellent inhibitory activity of DPP-4 with IC₅₀ values of 64.47?nM, 188.7?nM and 65.36?nM, respectively. Further studies revealed that compound 11 was potent in vivo hypoglycemic effect. The structure–activity relationships of these pyrimidinedione derivatives had been discussed, which would be useful for developing novel DPP-4 inhibitors as treating type 2 diabetes.     

Discovery of triazole-based uracil derivatives bearing amide moieties as novel dipeptidyl peptidase-IV inhibitors

Dipeptidyl peptidase-IV (DPP-4) is a validated target for T2DM treatment. We previously reported a novel series of triazole-based uracil derivatives bearing aliphatic carboxylic acids with potent DPP-4 inhibitory activities in vitro, but these compounds showed poor hypoglycemic effects in vivo. Herein we further optimized the triazole moiety by amidation of the carboxylic acid to improve in vivo activities. Two series of compounds 3a-f and 4a-g were designed and synthesized. By screening in DPP-4, compound 4c was identified as a potent DPP-4 inhibitor with the IC₅₀ value of 28.62 nM. Docking study revealed compound 4c has a favorable binding mode and interpreted the SAR of these analogs. DPP-8 and DPP-9 tests indicated compound 4c had excellent selectivity over DPP-8 and DPP-9. Further in vivo evaluations revealed that compound 4c showed more potent hypoglycemic activity than its corresponding carboxylic acid in ICR mice and dose-dependently reduced glucose levels in type 2 diabetic C57BL/6 mice. The overall results have shown that compound 4c could be a promising lead for further development of novel DPP-4 agents treating T2DM.