Deep learning driven de novo drug design based on gastric proton pump structures.

Existing drugs often suffer in their effectiveness due to detrimental side effects, low binding affinity or pharmacokinetic problems. This may be overcome by the development of distinct compounds. Here, we exploit the rich structural basis of drug-bound gastric proton pump to develop compounds with strong inhibitory potency, employing a combinatorial approach utilizing deep generative models for de novo drug design with organic synthesis and cryo-EM structural analysis. Candidate compounds that satisfy pharmacophores defined in the drug-bound proton pump structures, were designed in silico utilizing our deep generative models, a workflow termed Deep Quartet. Several candidates were synthesized and screened according to their inhibition potencies in vitro, and their binding poses were in turn identified by cryo-EM. Structures reaching up to 2.10 Å resolution allowed us to evaluate and re-design compound structures, heralding the most potent compound in this study, DQ-18 (N-methyl-4-((2-(benzyloxy)-5-chlorobenzyl)oxy)benzylamine), which shows a Ki value of 47.6 nM. Further high-resolution cryo-EM analysis at 2.08 Å resolution unambiguously determined the DQ-18 binding pose. Our integrated approach offers a framework for structure-based de novo drug development based on the desired pharmacophores within the protein structure.

Identification and characterization of receptors for ion transport peptide (ITP) and ITP-like (ITPL) in the silkworm Bombyx mori.

Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are specifically conserved in ecdysozoans. Many of the details of the molecular mechanisms by which crustacean hyperglycemic hormone family peptides exert pleiotropy remain to be elucidated, including characterization of their receptors. Here we identified three Bombyx mori orphan neuropeptide G protein-coupled receptors (BNGRs), BNGR-A2, -A24, and -A34, as receptors for ITP and ITPL (collectively referred to as ITPs). BNGR-A2 and -A34 and BNGR-A24 respond to recombinant ITPs, respectively, with EC50 values of 1.1-2.6 × 10(-8) M, when expressed in a heterologous expression system. These three candidate BNGRs are expressed at larval B. mori tissues targeted by ITPs, with cGMP elevation observed after exposure to recombinant ITPs. ITPs also increased the cGMP level in B. mori ovary-derived BmN cells via membrane-bound and soluble guanylyl cyclases. The simultaneous knockdown of bngr-A2 and -A34 significantly decreased the response of BmN cells to ITP, whereas knockdown of bngr-A24 led to decreased responses to ITPL. Conversely, transient expression of bngr-A24 potentiated the response of BmN cells to ITPL. An in vitro binding assay showed direct interaction between ITPs and heterologously expressed BNGRs in a ligand-receptor-specific manner. Taken together, these data demonstrate that BNGR-A2 and -A34 are ITP receptors and that BNGR-A24 is an ITPL receptor in B. mori.

Direct chemiluminescent enzyme immunoassay for atrial natriuretic peptide in mammalian plasma using a PEGylated antibody.

Atrial natriuretic peptide (ANP) is a peptide hormone that is synthesized and secreted by cardiac tissues and plays a pivotal role in maintaining cardiovascular homeostasis. Clinically, ANP is used as a marker of cardiovascular diseases, including heart failure. Although multiple ANP assays are currently available, a more sensitive assay is required for the direct measurement of plasma ANP where there is limited plasma availability, especially in mouse experiments. In the current study, we developed a plate-based sandwich chemiluminescent enzyme immunoassay for the measurement of plasma ANP in rats and mice without the need for prior extraction. To minimize nonspecific binding, we performed a single-step PEGylation procedure targeting the immobilized antibody, which markedly improved the assay's sensitivity and linearity. The linear range was 0.1 to 250 pM, and the minimum detection limit was 0.13 pM, 5-fold lower than the lowest value of the commercially available kits. ANP was directly measured in plasma samples without detectable cross-reactivity with B- and C-type natriuretic peptides. The accuracy of the assay was confirmed by spike recovery tests and dilution tests and by comparison with a conventional radioimmunoassay. Based on the species cross-reactivity, this assay can be used to measure human ANP.

Hexarelin treatment in male ghrelin knockout mice after myocardial infarction.

Both ghrelin and the synthetic analog hexarelin are reported to possess cardioprotective actions that are mainly exerted through different receptors. However, their effects on acute myocardial infarction have not been compared in vivo. This study aimed to clarify whether hexarelin treatment can compensate for ghrelin deficiency in ghrelin-knockout mice and to compare the effects of hexarelin (400 nmol/kg/d, sc) and equimolar ghrelin treatment after myocardial infarction. Myocardial infarction was produced by left coronary artery ligation in male ghrelin-knockout mice, which then received ghrelin, hexarelin, or vehicle treatment for 2 weeks. The mortality within 2 weeks was significantly lower in the hexarelin group (6.7%) and ghrelin group (14.3%) than in the vehicle group (50%) (P < .05). A comparison of cardiac function 2 weeks after infarction showed that in the ghrelin and hexarelin treatment groups, cardiac output was greater, whereas systolic function, represented by ejection fraction, and diastolic function, represented by dP/dt min (peak rate of pressure decline), were significantly superior compared with the vehicle group (P < .05). Hexarelin treatment was more effective than ghrelin treatment, as indicated by the ejection fraction, dP/dt max (peak rate of pressure rise), and dP/dt min. Telemetry recording and heart rate variability analysis demonstrated that sympathetic nervous activity was clearly suppressed in the hexarelin and ghrelin groups relative to the vehicle group. Our data demonstrated that hexarelin treatment can result in better heart function than ghrelin treatment 2 weeks after myocardial infarction in ghrelin-knockout mice, although both hormones have similar effects on heart rate variability and mortality.

Effects of starvation on brain short neuropeptide F-1, -2, and -3 levels and short neuropeptide F receptor expression levels of the silkworm, Bombyx mori.

In our previous report, we demonstrated the possibility that various regulatory neuropeptides influence feeding behavior in the silkworm, Bombyx mori. Among these feeding-related neuropeptides, short neuropeptide F (sNPF) exhibited feeding-accelerating activity when injected into B. mori larvae. Like other insect sNPFs, the deduced amino acid sequence of the cDNA encoding the sNPF precursor appears to produce multiple sNPF and sNPF-related peptides in B. mori. The presence of three sNPFs, sNPF-1, sNPF-2, and sNPF-3, in the brain of B. mori larvae was confirmed by direct MALDI-TOF mass spectrometric profiling. In addition, all three sNPFs are present in other larval ganglia. The presence of sNPF mRNA in the central nervous system (CNS) was also confirmed by Reverse transcription-polymerase chain reaction. Semi-quantitative analyses of sNPFs in the larval brain using matrix-assisted laser desorption ionization time-of-flight mass spectrometry further revealed that brain sNPF levels decrease in response to starvation, and that they recover with the resumption of feeding. These data suggest that sNPFs were depleted by the starvation process. Furthermore, food deprivation decreased the transcriptional levels of the sNPF receptor (BNGR-A10) in the brain and CNS, suggesting that the sNPF system is dependent on the feeding state of the insect and that the sNPF system may be linked to locomotor activity associated with foraging behavior. Since the injection of sNPFs accelerated the onset of feeding in B. mori larvae, we concluded that sNPFs are strongly related to feeding behavior. In addition, semi-quantitative MS analyses revealed that allatostatin, which is present in the larval brain, is also reduced in response to starvation, whereas the peptide level of Bommyosuppressin was not affected by different feeding states.

Crystallization and preliminary X-ray analysis of crustacean hyperglycaemic hormone from the kuruma prawn Marsupenaeus japonicus in its weakly active precursor form.

Crustacean hyperglycaemic hormone (CHH) plays a pivotal role in the regulation of glucose metabolism in crustaceans. Pej-SGP-I, one of the six known CHHs in the kuruma prawn Marsupenaeus japonicus, was heterologously expressed in Escherichia coli as an N-terminally His-tagged and Nus-tagged protein in its weakly active precursor form, Pej-SGP-I-Gly, which has an extra glycine residue at the C-terminus. The recombinant peptide was subjected to affinity purification, tag removal, further purification and crystallization by the sitting-drop vapour-diffusion method using NaCl as the main precipitant. The crystals diffracted to 1.95 Å resolution and the space group was assigned as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 40.19, b = 53.65, c = 53.63 Å. The Matthews coefficient (V(M) = 1.73 Å(3) Da(-1)) indicated that the crystal contained two Pej-SGP-I-Gly molecules per asymmetric unit, with a solvent content of 29.0%.

Effects of crustacean hyperglycemic hormone (CHH) on the transcript expression of carbohydrate metabolism-related enzyme genes in the kuruma prawn, Marsupenaeus japonicus.

Crustacean hyperglycemic hormone (CHH), a member of a neuropeptide family present only in arthropods, plays a pivotal role in the modulation of hemolymph glucose levels, molting, reproduction, and the stress response. Although it has been determined that hepatopancreas and muscle are the major tissues in which CHH regulates hyperglycemic activity, the molecular mechanism by which CHH regulates carbohydrate metabolism remains unclear. In this study, we analyzed the mRNA expression levels of enzymes involved in glycogen metabolism and gluconeogenesis in order to determine how CHH regulates hemolymph glucose levels. We first cloned cDNAs encoding four carbohydrate metabolism-related enzymes from the kuruma prawn, Marsupenaeus japonicus, glycogen phosphorylase (MjGP), glycogen synthase (MjGS), fructose 1,6-bisphosphatase (MjFBPase), and phosphoenolpyruvate carboxykinase (MjPEPCK). RT-PCR analysis showed that eyestalk ablation remarkably decreased MjGP and increased MjGS transcript levels in the hepatopancreas, but not in muscle. Considering the fact that various eyestalk factors, including MIH, are removed by eyestalk ablation, these results indicate that after eyestalk ablation the metabolic state proceeds towards glycogen accumulation in the specific tissues related to molting. In contrast, MjFBPase and MjPEPCK transcript levels were not significantly changed by eyestalk ablation, indicating that CHH and other eyestalk-derived factors might not induce gluconeogenesis. Quantitative real-time PCR analysis showed that exposure of hepatopancreas to recombinant CHH significantly changed the expression levels of MjGP and MjGS, but not MjFBPase and MjPEPCK. Collectively, these results indicate that CHH is involved in glycogen metabolism in hepatopancreas.

Identification of a second messenger of crustacean hyperglycemic hormone signaling pathway in the kuruma prawn Marsupenaeus japonicus.

To address the signaling pathway of crustacean hyperglycemic hormone (CHH) in the kuruma prawn Marsupenaeus japonicus, we measured the intracellular cAMP and cGMP levels in the hepatopancreas, one of the major target tissues of CHH. Removal of CHH-producing cells by bilateral eyestalk ablation decreased intracellular cGMP levels, while little effect on cAMP was observed in the tissue. Similarly, exposure to recombinant CHH significantly increased the cGMP level of the hepatopancreas in vitro, while little change was observed in cAMP. These results suggest that cGMP, rather than cAMP, mediates the CHH signal in hepatopancreas of M. japonicus.

A convenient method for preparation of biologically active recombinant CHH of the kuruma prawn, Marsupenaeus japonicus, using the bacterial expression system.

Crustacean hyperglycemic hormone (CHH) not only plays an important role in the modulation of hemolymph glucose level but also functions in other biological events including molting, reproduction and stress response. Of the six CHHs characterized in Marsupenaeus japonicus, an expression system for recombinant Pej-SGP-VII (rPej-SGP-VII-amide) has not yet been established. Here, we established a procedure using a Nus-tag for solubilization, thereby soluble and biologically active rPej-SGP-VII-amide could successfully be obtained by a simpler procedure than previous ones used for producing other recombinant Pej-SGPs (Pej-SGP-I, III and IV). It was found that rPej-SGP-VII-amide thus obtained had the correct arrangement of intramolecular disulfide bonds and helix-rich secondary structure. The established expression system for rPej-SGP-VII-amide may be applicable for the preparation of other recombinant CHHs.