We discovered a nonpeptidic compound, TAK-070, that inhibited BACE1, a rate-limiting protease for the generation of A␤ peptides that are considered causative for Alzheimer's disease (AD), in a noncompetitive manner. TAK-070 bound to full-length BACE1, but not to truncated BACE1 lacking the transmembrane domain. Short-term oral administration of TAK-070 decreased the brain levels of soluble A␤, increased that of neurotrophic sAPP␣ by ϳ20%, and normalized the behavioral impairments in cognitive tests in Tg2576 mice, an APP transgenic mouse model of AD. Six-month chronic treatment decreased cerebral A␤ deposition by ϳ60%, preserving the pharmacological efficacy on soluble A␤ and sAPP␣ levels. These results support the feasibility of BACE1 inhibition with a noncompetitive inhibitor as disease-modifying as well as symptomatic therapy for AD.

In our pursuit of developing a novel and potent potassium-competitive acid blocker (P-CAB), we synthesized pyrrole derivatives focusing on compounds with low log D and high ligand-lipophilicity efficiency (LLE) values. Among the compounds synthesized, the compound 13e exhibited potent H(+),K(+)-ATPase inhibitory activity and potent gastric acid secretion inhibitory action in vivo. Its maximum efficacy was more potent and its duration of action was much longer than those of proton pump inhibitors (PPIs). Therefore, compound 13e (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine fumarate, TAK-438) was selected as a drug candidate for the treatment of gastroesophageal reflux disease (GERD), peptic ulcer, and other acid-related diseases.

Metastin/kisspeptin is a 54 amino acid peptide ligand of the KISS1R receptor and is a critical regulator of GnRH secretion. The N-terminally truncated peptide, metastin(45-54), possesses a 10-fold higher receptor-binding affinity than full-length metastin and agonistic KISS1R activity but is rapidly inactivated in rodent plasma. We have developed a decapeptide analog [D-Tyr(45),D-Trp(47),azaGly(51),Arg(Me)(53)]metastin(45-54) with improved serum stability compared with metastin(45-54) but with decreased KISS1R agonistic activity. Amino acid replacements at positions 45-47 led to an enhancement of KISS1R agonistic activity and metabolic stability. N-terminal truncation resulted in a stable nonapeptide, [D-Tyr(46),D-Pya(4)(47),azaGly(51),Arg(Me)(53)]metastin(46-54), compound 26, which displayed KISS1R binding affinities comparable to metastin(45-54) and had improved serum stability. Compound 26 reduced plasma testosterone in male rats and is the first short-length metastin analog to possess testosterone suppressive activities. Compound 26 has led to the elucidation of investigational analogs TAK-683 and TAK-448, both of which have undergone clinical evaluation for hormone-dependent diseases such as prostate cancer.

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Modifications of metastin(45-54) produced peptide analogues with higher metabolic stability than metastin(45-54). N-terminally truncated nonapeptide 4 ([D-Tyr46,D-Pya(4)47,azaGly51,Arg(Me)53]metastin(46-54)) is a representative compound with both potent agonistic activity and metabolic stability. Although 4 had more potent testosterone-suppressant activity than metastin, it possessed physicochemical instability at pH 7 and insufficient in vivo activity. Instability at pH 7 was dependent upon Asn48 and Ser49; substitution of Ser49 with Thr49 reduced this instability and maintained KISS1 receptor agonistic activity. Furthermore, [D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54) (14) showed 2-fold greater [Ca2+]i-mobilizing activity than metastin(45-54) and an apparent increase in physicochemical stability. N-terminal acetylation of 14 resulted in the most potent analogue, 22 (Ac-[D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54)). With continuous administration, 22 possessed 10-50-fold more potent testosterone-suppressive activity in rats than 4. These results suggested that a controlled release of short-length KISS1 receptor agonists can suppress the hypothalamic-pituitary-gonadal axis and reduce testosterone levels. Compound 22 was selected for further preclinical evaluation for hormone-dependent diseases.

Elucidating the detailed mechanism of activation of membrane protein receptors and their ligand binding is essential for structure-based drug design. Membrane protein crystal structure analysis successfully aids in understanding these fundamental molecular interactions. However, protein crystal structure analysis of the G-protein-coupled receptor (GPCR) remains challenging, even for the class of GPCRs which have been included in the majority of structure analysis reports among membrane proteins, due to the substantial instability of these receptors when extracted from lipid bilayer membranes. It is known that increased thermostability tends to decrease conformational flexibility, which contributes to the generation of diffraction quality crystals. However, this is still not straightforward, and significant effort is required to identify thermostabilized mutants that are optimal for crystallography. To address this issue, a versatile screening platform based on a label-free ligand binding assay combined with transient overexpression in virus-like particles was developed. This platform was used to generate thermostabilized GPR40 [also known as free fatty acid receptor 1 (FFAR1)] for fasiglifam (TAK-875). This demonstrated that the thermostabilized mutant GPR40 (L42A/F88A/G103A/Y202F) was successfully used for crystal structure analysis.