Mitocryptide-2: Identification of Its Minimum Structure for Specific Activation of FPR2-Possible Receptor Switching from FPR2 to FPR1 by Its Physiological C-terminal Cleavages.

Mitocryptides are a novel family of endogenous neutrophil-activating peptides originating from various mitochondrial proteins. Mitocryptide-2 (MCT-2) is one of such neutrophil-activating peptides, and is produced as an N-formylated pentadecapeptide from mitochondrial cytochrome b. Although MCT-2 is a specific endogenous ligand for formyl peptide receptor 2 (FPR2), the chemical structure within MCT-2 that is responsible for FPR2 activation is still obscure. Here, we demonstrate that the N-terminal heptapeptide structure of MCT-2 with an N-formyl group is the minimum structure that specifically activates FPR2. Moreover, the receptor molecule for MCT-2 is suggested to be shifted from FPR2 to its homolog formyl peptide receptor 1 (FPR1) by the physiological cleavages of its C-terminus. Indeed, N-terminal derivatives of MCT-2 with seven amino acid residues or longer caused an increase of intracellular free Ca2+ concentration in HEK-293 cells expressing FPR2, but not in those expressing FPR1. Those MCT-2 derivatives also induced ß-hexosaminidase secretion in neutrophilic/granulocytic differentiated HL-60 cells via FPR2 activation. In contrast, MCT-2(1-4), an N-terminal tetrapeptide of MCT-2, specifically activated FPR1 to promote those functions. Moreover, MCT-2 was degraded in serum to produce MCT-2(1-4) over time. These findings suggest that MCT-2 is a novel critical factor that not only initiates innate immunity via the specific activation of FPR2, but also promotes delayed responses by the activation of FPR1, which may include resolution and tissue regeneration. The present results also strongly support the necessity of considering the exact chemical structures of activating factors for the investigation of innate immune responses.

Toxic Amyloid Tape: A Novel Mixed Antiparallel/Parallel ß-Sheet Structure Formed by Amyloid ß-Protein on GM1 Clusters.

The abnormal aggregation of amyloid ß-protein (Aß) is considered central in the pathogenesis of Alzheimer's disease. We focused on membrane-mediated amyloidogenesis and found that amyloid fibrils formed on monosialoganglioside GM1 clusters were more toxic than those formed in aqueous solution. In this study, we investigated the structure of the toxic fibrils by Aß-(1-40) in detail in comparison with less-toxic fibrils formed in aqueous solution. The less-toxic fibrils contain in-resister parallel ß-sheets, whereas the structure of the toxic fibrils is unknown. Atomic force microscopy revealed that the toxic fibrils had a flat, tape-like morphology composed of a single ß-sheet layer. Isotope-edited infrared spectroscopy indicated that almost the entire sequence of Aß is included in the ß-sheet. Chemical cross-linking experiments using Cys-substituted Aßs suggested that the fibrils mainly contained both in-resister parallel and two-residue-shifted antiparallel ß-sheet structures. Solid-state NMR experiments also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting plasmepsins - biochemical and structural insights.

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high-resolution crystal structures of PMII-KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI-10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use. DATABASE: Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.

Identification of Highly Potent Human Immunodeficiency Virus Type-1 Protease Inhibitors against Lopinavir and Darunavir Resistant Viruses from Allophenylnorstatine-Based Peptidomimetics with P2 Tetrahydrofuranylglycine.

The emergence of drug-resistant HIV from a widespread antiviral chemotherapy targeting HIV protease in the past decades is unavoidable and provides a challenge to develop alternative inhibitors. We synthesized a series of allophenylnorstatine-based peptidomimetics with various P3, P2, and P2́ moieties. The derivatives with P2 tetrahydrofuranylglycine (Thfg) were found to be potent against wild type HIV-1 protease and the virus, leading to a highly potent compound 21f (KNI-1657) against lopinavir/ritonavir- or darunavir-resistant strains. Co-crystal structures of 21f and the wild-type protease revealed numerous key hydrogen bonding interactions with Thfg. These results suggest that the strategy to design allophenylnorstatine-based peptidomimetics combined with Thfg residue would be promising for generating candidates to overcome multidrug resistance.

Generation of monoclonal antibodies against mitocryptide-2: toward a new strategy to investigate the biological roles of cryptides.

We recently identified a novel family of neutrophil-activating peptides including mitocryptide-1 and mitocryptide-2 (MCT-2) that are endogenously produced from various mitochondrial proteins. Among them, MCT-2 is an N-formylated pentadecapeptide derived from mitochondrial cytochrome b and is found to promote neutrophilic migration and phagocytosis efficiently. Signaling mechanisms of neutrophil activation by MCT-2 have been investigated at the cellular level, and MCT-2 has been demonstrated to be an endogenous specific ligand for formyl peptide receptor-2 (also referred to as formyl peptide receptor-like 1). It was also found that MCT-2 promoted neutrophilic functions via the activation of Gi2 proteins and phosphorylation of ERK1/2 consecutively. However, the physiological production, distribution, and functions of MCT-2 are not yet elucidated. Here, to investigate the roles of MCT-2 in vivo, we generated monoclonal antibodies (mAbs) against human MCT-2 (hMCT-2) that have two different characteristics. One mAb, NhM2A1, not only bound to the region of positions 10-15 of hMCT-2 but also recognized its C-terminal cleavage site that is presumably produced upon enzymatic hydrolysis of cytochrome b, indicating that NhM2A1 specifically interacts with hMCT-2 but not its parent protein. Moreover, we succeeded in acquiring a specific neutralizing mAb, NhM2A5, which blocks the bioactivities of hMCT-2. Specifically, NhM2A5 inhibited hMCT-2-induced ß-hexosaminidase release in neutrophilic/granulocytic differentiated HL-60 cells by binding to the region of positions 5-12 of hMCT-2. Functional analysis using obtained mAbs that specifically recognize hMCT-2 but not its parent protein, cytochrome b, and that neutralize bioactivities of hMCT-2 is expected to reveal the physiological roles of MCT-2, which are presently very difficult to investigate. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

New directions for protease inhibitors directed drug discovery.

Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, ß-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and ß-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016.

Mitochondrial protein-derived cryptides: Are endogenous N-formylated peptides including mitocryptide-2 components of mitochondrial damage-associated molecular patterns?

Recently, much attention has been paid to "nonclassical" bioactive peptides, which are fragmented peptides simultaneously produced during maturation and degradation of various functional proteins. We identified many fragmented peptides derived from various mitochondrial proteins including mitocryptide-1 and mitocryptide-2 that efficiently activate neutrophils. These endogenous, functionally active, fragmented peptides are referred to as "cryptides." Among them, mitocryptide-2 is an N-formylated cryptide cleaved from mitochondrial cytochrome b that is encoded in mitochondrial DNA (mtDNA). It is known that 13 proteins encoded in mtDNA are translated in mitochondria as N-formylated forms, suggesting the existence of endogenous N-formylated peptides other than mitocryptide-2. Here, we investigated the effects of N-formylated peptides presumably cleaved from mtDNA-encoded proteins other than cytochrome b on the functions of neutrophilic cells to elucidate possible regulation by endogenous N-formylated cryptides. Four N-formylated cryptides derived from cytochrome c oxidase subunit I and NADH dehydrogenase subunits 4, 5, and 6 among 12 peptides from mtDNA-encoded proteins efficiently induced not only migration but also ß-hexosaminidase release, which is an indicator of neutrophilic phagocytosis, in HL-60 cells differentiated into neutrophilic cells. These activities were comparable to or higher than those induced by mitocryptide-2. Although endogenous N-formylated peptides that are contained in mitochondrial damage-associated molecular patterns (DAMPs) have yet to be molecularly identified, they have been implicated in innate immunity. Thus, N-formylated cryptides including mitocryptide-2 are first-line candidates for the contents of mitochondrial DAMPs to promote innate immune responses. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 580-587, 2016.

Successful acquisition of a neutralizing monoclonal antibody against a novel neutrophil-activating peptide, mitocryptide-1.

Mitocryptide-1 (MCT-1) is a novel neutrophil-activating peptide derived from mitochondrial cytochrome c oxidase subunit VIII, and its physiological role and involvement in various diseases have not yet been elucidated. Generating neutralizing antibodies against the function of MCT-1 is of particular importance for investigating its physiological and pathophysiological roles, because MCT-1 is a fragmented peptide of its mother protein and hence it is very difficult to manipulate its expression level genetically without affecting expression of the mother protein. Here, we report the successful generation of a neutralizing monoclonal antibody (MAb) against MCT-1. This MAb, designated NM1B1, which specifically bound to the region of positions 9-22 of MCT-1, showed concentration-dependent inhibition of MCT-1-induced migration and ß-hexosaminidase release in neutrophilic/granulocytic differentiated HL-60 cells. Thus, NM1B1, as a neutralizing MAb against MCT-1, could elucidate not just the physiological regulatory mechanisms of MCT-1 but also its pathophysiological involvement in various inflammatory diseases in vivo.

Novel ß-amyloid aggregation inhibitors possessing a turn mimic.

Amyloid ß peptide, the main component of senile plaques found in the brain of Alzheimer disease (AD) patients, is a molecular target for AD therapeutic intervention. A number of potential AD therapeutics have been reported, including inhibitors of ß-secretase, γ-secretase, and Aß aggregation, and anti-amyloid agents, such as neprilysin, insulin degrading enzyme (IDE), and Aß antibodies. Recently, we reported potent small-sized ß-secretase (BACE1) inhibitors, which could serve as anti-AD drugs. However AD is a progressive disorder, where dementia symptoms gradually worsen over several decades, and therefore may require many years to get cured. One possible way to achieve a greater therapeutic effect is through simultaneous administration of multiple drugs, similar to those used in Highly Active Anti-Retroviral Therapy (HAART) used to treat AIDS. In order to overcome AD, we took a drug discovery approach to evaluate, novel ß-amyloid aggregation inhibitors. Previously, we reported that a tong-type compound possessing a turn mimic as the inhibitor of HIV-1 protease dimerization. Oligomerized amyloid ß peptides contain a turn structure within the molecule. Here, we designed and synthesized novel ß-amyloid aggregation inhibitors with a turn-mimic template, based on the turn conformer of the oligomerized amyloid ß peptides.

The zymogen of plasmepsin V from Plasmodium falciparum is enzymatically active.

Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies. Refolding conditions were optimized and zymogen was processed into a mature form via cleavage at the Asn80-Ala81 peptide bond. Mature plasmepsin V exhibited a pH optimum of 5.5-7.0 with Km and kcat of 4.6 µM and 0.24s(-1), respectively, at pH 6.0 using the substrate DABCYL-LNKRLLHETQ-E(EDANS). Furthermore, the prosegment of proplasmepsin V was shown to be nonessential for refolding and inhibition. Unexpectedly, unprocessed proplasmepsin V was enzymatically active with slightly reduced substrate affinity (∼ 2-fold), and similar pH optimum as well as turnover compared to the mature form. Both zymogenic and mature plasmepsin V were partially inhibited by pepstatin A as well as several KNI aspartic protease inhibitors while certain metals strongly inhibited activity. Overall, the present study provides the first report on the nonessentiality of the prosegment for plasmepsin V folding and activity, and therefore, subsequent characterization of its structure-function relationships of both zymogen and mature forms in the development of novel inhibitors with potential antimalarial activities is warranted.

Bacteria of the candidate phylum TM7 are prevalent in acidophilic nitrifying sequencing-batch reactors.

Laboratory-scale acidophilic nitrifying sequencing-batch reactors (ANSBRs) were constructed by seeding with sewage-activated sludge and cultivating with ammonium-containing acidic mineral medium (pH 4.0) with or without a trace amount of yeast extract. In every batch cycle, the pH varied between 2.7 and 4.0, and ammonium was completely converted to nitrate. Attempts to detect nitrifying functional genes in the fully acclimated ANSBRs by PCR with previously designed primers mostly gave negative results. 16S rRNA gene-targeted PCR and a subsequent denaturating gradient gel electrophoresis analysis revealed that a marked change occurred in the bacterial community during the overall period of operation, in which members of the candidate phylum TM7 and the class Gammaproteobacteria became predominant at the fully acclimated stage. This result was fully supported by a 16S rRNA gene clone library analysis, as the major phylogenetic groups of clones detected (>5% of the total) were TM7 (33%), Gammaproteobacteria (37%), Actinobacteria (10%), and Alphaproteobacteria (8%). Fluorescence in situ hybridization with specific probes also demonstrated the prevalence of TM7 bacteria and Gammaproteobacteria. These results suggest that previously unknown nitrifying microorganisms may play a major role in ANSBRs; however, the ecophysiological significance of the TM7 bacteria predominating in this process remains unclear.

Non-pretreated O-acyl isopeptide of amyloid ß peptide 1-42 is monomeric with a random coil structure but starts to aggregate in a concentration-dependent manner.

An isopeptide of amyloid ß peptide 1-42 (isoAß42) was considered as a non-aggregative precursor molecule for the highly aggregative Aß42. It has been applied to biological studies after several pretreatments. Here we report that isoAß42 is monomeric with a random coil structure at 40 µM without any pretreatment. But we also found that isoAß42 retains a slight aggregative nature, which is significantly weaker than that of the native Aß42.

O-acyl isopeptide method: development of an O-acyl isodipeptide unit for Boc SPPS and its application to the synthesis of Aß1-42 isopeptide.

The O-acyl isopeptide method was developed for the efficient preparation of difficult sequence-containing peptide. Furthermore, development of the O-acyl isodipeptide unit for Fmoc chemistry simplified its synthetic procedure by solid-phase peptide synthesis. Here, we report a novel isodipeptide unit for Boc chemistry, and the unit was successfully applied to the synthesis of amyloid ß peptide. Combination of Boc chemistry and the isodipeptide unit would be an effective method for the synthesis of many difficult peptides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Optimization of plasmepsin inhibitor by focusing on similar structural feature with chloroquine to avoid drug-resistant mechanism of Plasmodium falciparum.

The plasmepsins are specific aspartic proteases of the malaria parasite and a potential target for developing new antimalarial agents. Our previously reported peptidomimetic plasmepsin inhibitor with modified 2-aminoethylamino substituent, KNI-10740, was tested against chloroquine sensitive Plasmodium falciparum, D6, to be highly potent, however, the inhibitor exhibited about 5 times less activity against multi-drug resistant parasite (TM91C235). We hypothesized the potency reduction resulted from structural similarity between 2-aminoethylamino substituent of KNI-10740 and chloroquine. Then, we modified the moiety and finally identified compound 15d (KNI-10823), that could avoid drug-resistant mechanism of TM91C235 strain.

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, ß-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 µM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.

Structure-activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P(2) position.

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aß production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood-brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P2 position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.

A fluorogenic probe for γ-glutamyl cyclotransferase: application of an enzyme-triggered O-to-N acyl migration-type reaction.

Light it up: human chromosome 7 ORF 24, a tumor-related protein, has been identified as a γ-glutamyl cyclotransferase (GGCT) in the glutathione homeostasis cycle. The singular substrate preference of the enzyme has hampered chemical probe development, and no fluorogenic probe has been reported. Here we report the first fluorogenic dipeptide probe, LISA-4, which should contribute toward further understanding of GGCT.

A new class of aggregation inhibitor of amyloid-ß peptide based on an O-acyl isopeptide.

Inhibition of amyloid ß peptide (Aß) aggregation is a potential therapeutic approach to treat Alzheimer's disease. We report that an O-acyl isopeptide of Aß1-42 (1) containing an ester bond at the Gly(25)-Ser(26) moiety inhibits Aß1-42 fibril formation at equimolar ratio. Inhibitory activity was retained by an N-Me-ß-Ala(26) derivative (2), in which the ester of 1 was replaced with N-methyl amide to improve chemical stability at physiological pH. Inhibition was verified by fluorescence anisotropy, Western blot, and atomic force microscopy. This report suggests a new class of Aß aggregation inhibitor based on modification of Aß1-42 at Gly(25)-Ser(26).

Novel BACE1 inhibitors with a non-acidic heterocycle at the P1' position.

We have reported potent peptidic and non-peptidic BACE1 inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. However, our potent inhibitors possess a tetrazole ring at the P1' position. It is desirable that central nervous system (CNS) drugs do not possess an acidic moiety. In this study, we synthesized non-acidic BACE1 inhibitors with heterocyclic derivatives at the P1' position. KMI-1764 (27) exhibited potent inhibitory activity (IC50=27nM). Interestingly, these non-acidic inhibitors tended to follow the quantitative structure-activity relationship (QSAR) equation and interacted with BACE1-Arg235 in the binding model.