Discovery of FXR/PPARγ dual partial agonist.

Farnesoid X receptor (FXR) and peroxisome proliferator-activated receptor (PPAR)γ are nuclear receptor 1 superfamily of transcription factors. FXR and PPARγ agonists have been individually investigated in clinical trial of anti-diabetic agents in the patients with nonalcoholic fatty liver disease (NAFLD). Regarding recent agonist development, the partial agonists for FXR and PPARγ are drawing attention from the standpoint of avoiding overactive responses caused by full agonists. In this article, we report that 18 with a benzimidazole scaffold possesses FXR/PPARγ dual partial agonistic activity. In addition, 18 shares the ability to reduce cyclin-dependent kinase 5-mediated phosphorylation of PPARγ-Ser273 and the metabolic stability in mouse liver microsome assay. To date, there are no published reports on FXR/PPARγ dual partial agonists with biological profiles similar to 18. Thus, the analog would be a feasible candidate as an unprecedented approach to NAFLD associated with type 2 diabetes mellitus.

Design and identification of a new farnesoid X receptor (FXR) partial agonist by computational structure-activity relationship analysis: Ligand-induced H8 helix fluctuation in the ligand-binding domain of FXR may lead to partial agonism.

Farnesoid X receptor (FXR) controls gene-expression relevant to various diseases including nonalcoholic steatohepatitis and has become a drug target to regulate metabolic aberrations. However, some side effects of FXR agonists reported in clinical development such as an increase in blood cholesterol levels incentivize the development of partial agonists to minimize side effects. In this study, to identify a new partial agonist, we analyzed the computational structure-activity relationship (SAR) of FXR agonists previously developed in our laboratories using molecular dynamics simulations. SAR analysis showed that fluctuations in the H8 helix, by ligand binding, of the ligand-binding domain (LBD) of FXR may influence agonistic activity. Based on this observation, 6 was newly designed as a partial agonist and synthesized. As a result of biological evaluations, 6 showed weak agonistic activity (40.0% relative agonistic activity to the full-agonist GW4064) and a potent EC50 value (55.5 nM). The successful identification of the new potent partial agonist 6 suggested that helix fluctuation in the LBD induced by ligands could be one way to develop partial agonists.

N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) agonists accompanying prominent selectivity against vitamin D receptor (VDR).

As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. With respect to the bone metabolism, FXR positively regulates bone metabolism through both bone formation and resorption of the bone remodeling pathways. Some of FXR agonists possessing isoxazole moiety are undergoing clinical trials for the treatment of non-alcoholic steatohepatitis. To date, therefore, the activation of FXR leads to considerable interest in FXR as potential therapeutic targets. We have identified a series of nonsteroidal FXR agonists bearing N1-methyl benzimidazole and isoxazole moieties that are bridged with aromatic derivatives. They showed affinity to FXR, but also weak affinity toward the vitamin D receptor (VDR) that involves regulation of calcium and phosphate homeostasis and is activated by bile acids. The deployment of FXR agonists without activity against VDR as off-target is therefore crucial in the development of FXR ligands. Our efforts focusing on increasing the agonist properties towards FXR led to the discovery of 19, which activates FXR at and below nanomolar levels (EC50 = 26.5 ± 10.5 nM TR-FRET and 0.8 ± 0.2 nM luciferase, respectively) and functions as a FXR agonist: the affinity toward FXR over eight nuclear receptors, including VDR [IC50 (VDR) / EC50 (FXR) > 5000] and TGR5, effects FXR target genes, and activates bone morphogenetic protein-2-induced differentiation of mouse bone marrow-derived mesenchymal stem cell-like ST2 cells into osteoblast.

Identification of potent farnesoid X receptor (FXR) antagonist showing favorable PK profile and distribution toward target tissues: Comprehensive understanding of structure-activity relationship of FXR antagonists.

Antagonizing transcriptional activity of farnesoid X receptor (FXR) in the intestine has been reported as an effective means for the treatment of nonalcoholic fatty liver disease, type 2 diabetes and obesity. We describe herein that the building blocks necessary to maintain the antagonism of our chemotype were investigated in order to modulate in vivo pharmacokinetic behavior and the tissue distribution without blunting the activity against FXR. A comprehensive understanding of the structure-activity relationship led to analog 30, which is superior to 12 in terms of its pharmacokinetic profiles by oral administration and its tissue distribution toward target tissues (liver and ileum) in rats while preserving the in vitro activity of 12 against FXR. Thus, 30 should be a candidate compound to investigate the effects of inhibiting FXR activity while simultaneously improving the outcome of nonalcoholic fatty liver disease, type 2 diabetes and obesity.

Synthesis of Novel Farnesoid X Receptor Agonists and Validation of Their Efficacy in Activating Differentiation of Mouse Bone Marrow-Derived Mesenchymal Stem Cells into Osteoblasts.

The modulators of farnesoid X receptor (FXR), a bile acid receptor, regulate various biological processes including bile acid metabolism, and are associated with the control of fatty liver and osteoporosis. Thus, the control of FXR activity and development of FXR modulators are critical not only for research, but also for clinical application. In this study, we synthesized novel FXR agonists 1-4 possessing isoxazole and N-substituted benzimidazole moieties, and compared their effects on osteoblast differentiation with the known FXR agonists, chenodeoxycholic acid and a synthetic compound, GW4064. Two (3 and 4) of the four novel FXR agonists 1-4 showed high specificities for FXR. Computer-assisted modeling suggested that the binding of the FXR agonist 3 with ligand binding domain of FXR was similar to GW4064. FXR was expressed in mouse bone marrow-derived mesenchymal stem cell (MSC)-like ST2 cells (ST-2 MSCs). The FXR agonists activated the BMP-2-induced differentiation of ST-2 MSCs into osteoblasts and enhanced the expression of RUNX2. Moreover, the potency of the FXR agonist 3 was comparable to GW4064 in promoting osteoblast differentiation of ST-2 MSCs. These results indicate that FXR activation enhanced the BMP-2-induced differentiation of MSCs into osteoblasts through activating RUNX2 expression. FXR could be a potential therapeutic target for the treatment of bone diseases such as osteoporosis.

Discovery and optimization of benzimidazole derivatives as a novel chemotype of farnesoid X receptor (FXR) antagonists.

We describe here a novel chemotype with substituted benzimidazole scaffold for nonsteroidal farnesoid X receptor (FXR) antagonists starting from the identification of a screening hit, BB-4. Structure diversity in four regions A-D of BB-4 or 1 is discussed. In particular, regions A and C had an effect on an antagonism against FXR as demonstrated by the derivatives represented by 7 and 15, respectively. Thus, compound 19 arising from the combination of regions A and C underscored an important fact on antagonism against FXR, also showing the reduced small heterodimer partner and the increased cholesterol 7α-hydroxylase expression levels.

Plasmin inhibitors with hydrophobic amino acid-based linker between hydantoin moiety and benzimidazole scaffold enhance inhibitory activity.

In this letter we report the design and synthesis of a series of plasmin inhibitors, which share the amino acid-based linker with limited free rotation between the hydantoin moiety and the benzimidazole scaffold. Our studies led to potent plasmin inhibitors and yielded important new insights into their structure-activity relationship for binding to the active site of plasmin.

Active site-directed plasmin inhibitors: Extension on the P2 residue.

Based on the structure of YO-2 [N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr(O-picolyl)-NH-octyl], active site-directed plasmin (Plm) inhibitors were explored. The picolyl moiety in the Tyr(O-picolyl) residue (namely, the P2 residue) was replaced with smaller or larger groups, such as hydrogen, tert-butyl, benzyl, (2-naphthyl)methyl, and (quinolin-2-yl)methyl. Those efforts produced compound 17 {N-(trans-4-aminomethylcyclohexanecarbonyl)-l-Tyr[O-(quinolin-2-yl)methyl]-NH-octyl} [IC50=0.22 and 77µM for Plm and urokinase (UK), respectively], which showed not only 2.4-fold greater Plm inhibition than YO-2, but also an improvement in selectivity (Plm/UK) by 35-fold. The docking experiments of the Plm-17 complexes disclosed that the amino group of the tranexamyl moiety interacted with the side-chain of Asp753 which formed S1 site.

Novel type of plasmin inhibitors: providing insight into P4 moiety and alternative scaffold to pyrrolopyrimidine.

Here we report a series of plasmin inhibitors which were originally derived from the parent structure of 1 and 2. Our efforts focused on the optimization of the P4 moiety of 2 and on the quest of alternative scaffold to pyrrolopyrimidine in the parent compounds. The results of the former gave us pivotal information on the further optimization of the P4 moiety in plasmin inhibitors and those of the latter revealed that appropriate moieties extending from the benzimidazole scaffold engaged with S4 pocket in the active site of plasmin.

Pyrrolopyrimidine-inhibitors with hydantoin moiety as spacer can explore P4/S4 interaction on plasmin.

In the development of plasmin inhibitors, a novel chemotype, pyrrolopyrimidine scaffold possessing two motifs, a hydantoin-containing P4 moiety and a warhead-containing P1 moiety, is uncovered. A unique feature of the new line of the plasmin inhibitors is that the interaction between the plasmin inhibitors and key subsites in plasmin can be controlled by a spacer like hydantoin. The application of the novel chemotype is demonstrated by 1n and provides further evidence on the importance of hydantoin as the spacer.

Synthesis and evaluation of tripeptidic plasmin inhibitors with nitrile as warhead.

Plasmin is best known as the key molecule in the fibrinolytic system, which is critical for clot lysis and can initiate matrix metalloproteinase (MMP) activation cascade. Along with MMP, plasmin is suggested to be involved in physiological processes that are linked to the risk of carcinoma formation. Plasmin inhibitors could be perceived as a promising new principle in the treatment of diseases triggered by plasmin. On the basis of the peptidic sequence derived from the synthetic plasmin substrate, a series of peptidic plasmin inhibitors possessing nitrile as warhead were prepared and evaluated for their inhibitory activities against plasmin and other serine proteases, plasma kallikrein and urokinase. The most potent peptidic inhibitors with the nitrile warhead exhibit the potency toward plasmin (IC(50) = 7.7-11 µM) and are characterized by their selectivity profile against plasma kallikrein and urokinase. The results and molecular modeling of the peptidic inhibitor complexed with plasmin reveal that the P2 residue makes favorable contacts with the open binding pocket comprising the S2 and S3 subsites of plasmin.

Predicting subsite interactions of plasmin with substrates and inhibitors through computational docking analysis.

Plasmin plays important roles in various physiological systems. The identification of inhibitors controlling its regulation represents a promising drug-discovery challenge. To develop selective inhibitors of plasmin, structural information of the binding modes is crucial. Here, a computational docking study was conducted to provide structural insight into plasmin subsite interactions with substrates/inhibitors. Predicted binding modes of two peptide-substrates (D/L-Ile-Phe-Lys), and potent and weak inhibitors (YO-2 and PKSI-527) suggested non-prime and prime subsite interactions relevant to recognition by plasmin. Predicted binding modes also correlated well with the experimental structure-activity relationships for plasmin substrates/inhibitors, namely the differences of K(M) values between the D- and L-peptide-substrates and inhibitory potencies of YO-2 and PKSI-527. In particular, interaction observed at a hydrophobic pocket near S2 and at a tunnel-shaped hydrophobic S1' was strongly suggested to be significantly involved in tight binding of inhibitors to plasmin. Our present findings may aid in the design of potent and selective plasmin inhibitors.

Identification of novel plasmin inhibitors possessing nitrile moiety as warhead.

Lysine-nitrile derivatives having a trisubstituted benzene, which belongs to a new chemical class, were prepared and tested for inhibitory activities against plasmin and the highly homologous plasma kallikrein and urokinase. The use of the novel chemotype in the development of plasmin inhibitors has been demonstrated by derivatives of compound 9.

Discovery of Orally Active and Nonsteroidal Farnesoid X Receptor (FXR) Antagonist with Propensity for Accumulation and Responsiveness in Ileum.

We describe the discovery of analog 15 (FLG249), which is an orally active and nonsteroidal farnesoid X receptor (FXR) antagonist in mice with unique profiles, such as a propensity for ileum distribution and the significant control in the expression level of three FXR target genes in mouse ileum. Key design features incorporated in 15 were the introduction of metabolically stable groups in potent and metabolically labile antagonist 9. Our pursuit ultimately identified FXR antagonist 15, which has enabled its assessment in a drug discovery program.

Species differences between human and rat in the substrate specificity of cathepsin K.

Cathepsin K is known to play an important role in bone resorption, and it has the P2 specificity for proline. Rat cathepsin K has 88% identity with the human enzyme. However, it has been reported that its enzymatic activity for a Cbz-Leu-Arg-MCA substrate is lower than that of human cathepsin K, and that the rat enzyme is not well inhibited by human cathepsin K inhibitors. For this study, we prepared recombinant enzyme to investigate the substrate specificity of rat cathepsin K. Cleavage experiments using the fragment of type I collagen and peptidic libraries demonstrated that rat cathepsin K preferentially hydrolyses the substrates at the P2 Hyp position. Comparison of the S2 site between rat and human cathepsin K sequences indicated that two S2 residues at Ser134 and Val160 in rat are varied to Ala and Leu, respectively, in the human enzyme. Cleavage experiments using two single mutants, S134A and V160L, and one double mutant, S134A/V160L, of rat cathepsin K showed that all the rat mutants lost the P2 Hyp specificity. The information obtained from our comparative studies on rat and human cathepsin K should make a significant impact on developing specific inhibitors of human cathepsin K since rat is usually used as test species.

New chemotypes for cathepsin K inhibitors.

Cyano pyrimidine acetylene and cyano pyrimidine t-amine, which belong to a new chemical class, were prepared and tested for inhibitory activities against cathepsin K and the highly homologous cathepsins L and S. The use of novel chemotypes in the development of cathepsin K inhibitors has been demonstrated by derivatives of compounds 1 and 8.

Discovery of selective and nonpeptidic cathepsin S inhibitors.

Nonpeptidic, selective, and potent cathepsin S inhibitors were derived from an in-house pyrrolopyrimidine cathepsin K inhibitor by modification of the P2 and P3 moieties. The pyrrolopyrimidine-based inhibitors show nanomolar inhibition of cathepsin S with over 100-fold selectivity against other cysteine proteases, including cathepsin K and L. Some of the inhibitors showed cellular activities in mouse splenocytes as well as oral bioavailabilities in rats.

Overcoming hERG issues for brain-penetrating cathepsin S inhibitors: 2-cyanopyrimidines. Part 2.

We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K(+) channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.

Nonacidic Chemotype Possessing N-Acylated Piperidine Moiety as Potent Farnesoid X Receptor (FXR) Antagonists.

Farnesoid X receptor (FXR) plays a major role in the control of cholesterol metabolism. Antagonizing transcriptional activity of FXR is an effective means to treat the relevant metabolic syndrome. Some of antagonists so far have the charged functions; however, they may negatively affect the pharmacokinetics. We describe herein a structure-activity relationship (SAR) exploration of nonacidic FXR antagonist 6 focusing on two regions in the structure and biological evaluation of nonacidic 10 with the characteristic N-acylated piperidine group obtained from SAR studies. As the robust affinity to FXR is feasible with our nonacidic analogue, 10 is among the most promising candidates for in vivo testing.

2-Cyano-pyrimidines: a new chemotype for inhibitors of the cysteine protease cathepsin K.

Starting from the purine lead structure 1, a new series of cathepsin K inhibitors based on a pyrimidine scaffold have been explored. Investigations of P3 and P2 substituents based on molecular modeling suggestions resulted in potent cathepsin K inhibitors with an improved selectivity profile over other cathepsins.