Introduction of C-alkyl branches to L-iminosugars changes their active site binding orientation.

L-ido-Deoxynojirimycin (L-ido-DNJ) itself showed no affinity for human lysosomal acid α-glucosidase (GAA), whereas 5-C-methyl-L-ido-DNJ showed a strong affinity for GAA, comparable to the glucose analog DNJ, with a Ki value of 0.060 µM. This excellent affinity for GAA and enzyme stabilization was observed only when methyl and ethyl groups were introduced. Docking simulation analysis revealed that the alkyl chains of 5-C-alkyl-L-ido-DNJs were stored in three different pockets, depending on their length, thereby the molecular orientation was changed. Comparison of the binding poses of DNJ and 5-C-methyl-L-ido-DNJ showed that they formed a common ionic interaction with Asp404, Asp518, and Asp616, but both the binding orientation and the distance between the ligand and each amino acid residue were different. 5-C-Methyl-L-ido-DNJ dose-dependently increased intracellular GAA activity in Pompe patient fibroblasts with the M519V mutation and also promoted enzyme transport to lysosomes. This study provides the first example of a strategy to design high-affinity ligands by introducing alkyl branches into rare sugars and L-sugar-type iminosugars to change the orientation of binding.

Synthesis and Evaluation of Habiterpenol Analogs.

Habiterpenol is a G2 checkpoint inhibitor isolated from the culture broth of Phytohabitans sp. 3787_5. Here, we report the synthesis of new habiterpenol analogs through the total synthesis process of habiterpenol and evaluating the analogs for G2 checkpoint inhibitory activity. We investigated two different synthetic approaches for total synthesis, with intramolecular conjugate addition and Ti(III)-mediated radical cyclization as key reactions. Although the former was unsuccessful, the latter reaction facilitated stereoselective total synthesis and determination of the absolute configuration of habiterpenol. The extension of these chemistries to a structure-activity relationship (SAR) study gave new habiterpenol analogs, which could not be derived from natural habiterpenol and only be synthesized by applying the total synthesis. Therefore, this study provides important insights into SAR studies of habiterpenol.

Analysis on the Substrate Specificity of Recombinant Human Acyl-CoA Synthetase ACSL4 Variants.

Acyl-CoA synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs. ACSL4 is an ACSL isozyme with a strong preference for arachidonic acid (AA) and has been hypothesized to modulate the metabolic fates of AA. There are two ACSL4 splice variants: ACSL4V1, which is the more abundant transcript, and ACSL4V2, which is believed to be restricted to the brain. In the present study, we expressed recombinant human ACSL4V1 and V2 in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system and then partially purified both variants by cobalt affinity column chromatography. We then established a novel ACSL assay system with LC-MS/MS, which is highly sensitive and applicable to various kinds of fatty acids, and used it to investigate the substrate specificity of recombinant human ACSL4V1 and V2. The results showed that both ACSL4 variants preferred various kinds of highly unsaturated fatty acids (HUFAs), including docosahexaenoic acid (DHA), adrenic acid (docosatetraenoic acid) and eicosapentaenoic acid (EPA), as well as AA as a substrate. Moreover, our kinetic studies revealed that the two variants had similar relative affinities for AA, EPA and DHA but different reaction rates for each HUFA. These results confirmed the importance of both of ACSL4 variants in the maintenance of membrane phospholipids bearing HUFAs. Structural analysis of these variants might reveal the molecular mechanism by which they maintain membrane phospholipids bearing HUFAs.

Reverse N-Substituted Hydroxamic Acid Derivatives of Fosmidomycin Target a Previously Unknown Subpocket of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase (DXR).

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

The stability of NPM1 oligomers regulated by acidic disordered regions controls the quality of liquid droplets.

The nucleolus is a membrane-less nuclear body that typically forms through the process of liquid-liquid phase separation (LLPS) involving its components. NPM1 drives LLPS within the nucleolus and its oligomer formation and inter-oligomer interactions play a cooperative role in inducing LLPS. However, the molecular mechanism underlaying the regulation of liquid droplet quality formed by NPM1 remains poorly understood. In this study, we demonstrate that the N-terminal and central acidic residues within the intrinsically disordered regions (IDR) of NPM1 contribute to attenuating oligomer stability, although differences in the oligomer stability were observed only under stringent conditions. Furthermore, the impact of the IDRs is augmented by an increase in net negative charges resulting from phosphorylation within the IDRs. Significantly, we observed an increase in fluidity of liquid droplets formed by NPM1 with decreased oligomer stability. These results indicate that the difference in oligomer stability only observed biochemically under stringent conditions has a significant impact on liquid droplet quality formed by NPM1. Our findings provide new mechanistic insights into the regulation of nucleolar dynamics during the cell cycle.

Design and Pharmacological Chaperone Effects of N-(4'-Phenylbutyl)-DAB Derivatives Targeting the Lipophilic Pocket of Lysosomal Acid α-Glucosidase.

This study provides the first example of a strategy to design a practical ligand toward lysosomal acid α-glucosidase (GAA) focusing on N-alkyl derivatives of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB). The optimized N-4'-(p-trifluoromethylphenyl)butyl-DAB (5g) showed a Ki value of 0.73 µM, which was 353-fold higher affinity than N-butyl-DAB (3f) without a terminal phenyl group. Docking analysis showed that the phenyl part of 5g was accommodated in a lipophilic pocket. Furthermore, the p-trifluoromethyl group effectively suppresses the fluctuation of the phenyl group, allowing it to produce a stable bonding form with GAA. 5g increased the midpoint of the protein's protein denaturation temperature (Tm) by 6.6 °C above that in the absence of the ligand and acted as a "thermodynamic stabilizer" to improve the thermal stability of rhGAA. 5g dose-dependently increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation; its effect was comparable to that of DNJ, which is under clinical trials.

Structure of the His269Arg mutant of the rat aldose reductase-like protein AKR1B14 complexed with NADPH.

Rat aldose reductase-like protein (AKR1B14) is an orthologue of mouse vas deferens protein (AKR1B7) and plays roles in the detoxification of reactive aldehydes and synthesis of prostaglandin F(2α). Here, the 1.87 Å resolution crystal structure of the His269Arg mutant of AKR1B14 complexed with NADPH is described and shows that the negatively charged 2'-phosphate group of the coenzyme forms an ionic interaction with the positively charged guanidinium group of Arg269 that is also observed in the human aldose reductase (AKR1B1) structure. Previous experiments on the site-directed mutagenesis of His269 to Arg, Phe and Met revealed fourfold, sevenfold and 127-fold increases in the K(m) for NADPH, respectively, which are in agreement with the present molecular-modelling and X-ray crystallographic studies. This is the first tertiary structure of a mutant form of this AKR1B7 orthologue to be reported in order to investigate the structure-function relationship of the nonconserved His269 and its role in coenzyme binding.

Over 40 Years of Fosmidomycin Drug Research: A Comprehensive Review and Future Opportunities.

To address the continued rise of multi-drug-resistant microorganisms, the development of novel drugs with new modes of action is urgently required. While humans biosynthesize the essential isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the established mevalonate pathway, pathogenic protozoa and certain pathogenic eubacteria use the less well-known methylerythritol phosphate pathway for this purpose. Important pathogens using the MEP pathway are, for example, Plasmodium falciparum, Mycobacterium tuberculosis, Pseudomonas aeruginosa and Escherichia coli. The enzymes of that pathway are targets for antiinfective drugs that are exempt from target-related toxicity. 2C-Methyl-D-erythritol 4-phosphate (MEP), the second enzyme of the non-mevalonate pathway, has been established as the molecular target of fosmidomycin, an antibiotic that has so far failed to be approved as an anti-infective drug. This review describes the development and anti-infective properties of a wide range of fosmidomycin derivatives synthesized over the last four decades. Here we discuss the DXR inhibitor pharmacophore, which comprises a metal-binding group, a phosphate or phosphonate moiety and a connecting linker. Furthermore, non-fosmidomycin-based DXRi, bisubstrate inhibitors and several prodrug concepts are described. A comprehensive structure-activity relationship (SAR) of nearly all inhibitor types is presented and some novel opportunities for further drug development of DXR inhibitors are discussed.

5-C-Branched Deoxynojirimycin: Strategy for Designing a 1-Deoxynojirimycin-Based Pharmacological Chaperone with a Nanomolar Affinity for Pompe Disease.

In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 µM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 µM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

Crystallization and preliminary X-ray crystallographic analysis of human autotaxin.

Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0 Šresolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9 Å, ß = 122.6°.

Novel angular naphthopyrone formation by Arp1p dehydratase involved in Aspergillus fumigatus melanin biosynthesis.

Conidial pigment is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. The biosynthetic gene cluster for 1,8-dihydroxynaphthalene (DHN)-melanin in A. fumigatus consists of six genes, alb1, ayg1, arp1, arp2, abr1 and abr2. In contrast to black DHN-melanin fungi such as Magnaporthe grisea, the polyketide synthase Alb1p in A. fumigatus produces naphthopyrone YWA1 instead of 1,3,6,8-THN (T4HN) and YWA1 is converted to T4HN by Ayg1p. The yeast transformant expressing Alb1p and Arp1p dehydratase produced an unknown compound which was identified to be a novel angular naphthopyrone named YWA3 formed from YWA1. In addition, the amount of YWA3 produced was much more than that of YWA2 formed by non-enzymatic dehydration from YWA1. To further analyse the reaction in vitro, Arp1p was overexpressed in E. coli and purified. Kinetic analysis revealed Km value of Arp1p for YWA1 to be 41 µM which is comparable with that of Ayg1p for YWA1 in conversion to T4HN. The complex structure modelling well explained the mechanism of YWA3 generation by the dehydration of angular YWA1 by Arp1p. These results indicated the possibility that polymerization of angular naphthopyrone YWA3 but not YWA2 could be involved in the characteristic bluish-green conidial pigmentation of A. fumigatus.

Structural basis for an exceptionally strong preference for asparagine residue at the S2 subsite of Stenotrophomonas maltophilia dipeptidyl peptidase 7.

The emergence of drug-resistant bacteria has become a major problem worldwide. Bacterial dipeptidyl peptidases 7 and 11 (DPP7s and DPP11s), belonging to the family-S46 peptidases, are important enzymes for bacterial growth and are not present in mammals. Therefore, specific inhibitors for these peptidases are promising as potential antibiotics. While the molecular mechanisms underlining strict specificity at the S1 subsite of S46 peptidases have been well studied, those of relatively broad preference at the S2 subsite of these peptidases are unknown. In this study, we performed structural and biochemical analyses on DPP7 from Stenotrophomonas maltophilia (SmDPP7). SmDPP7 showed preference for the accommodation of hydrophobic amino acids at the S2 subsite in general, but as an exception, also for asparagine, a hydrophilic amino acid. Structural analyses of SmDPP7 revealed that this exceptional preference to asparagine is caused by a hydrogen bonding network at the bottom of the S2 subsite. The residues in the S2 subsite are well conserved among S46 peptidases as compared with those in the S1 subsite. We expect that our findings will contribute toward the development of a universal inhibitor of S46 peptidases.

Crystallization and preliminary X-ray crystallographic study of 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Plasmodium falciparum.

The nonmevalonate pathway of isoprenoid biosynthesis present in Plasmodium falciparum is known to be an effective target for antimalarial drugs. The second enzyme of the nonmevalonate pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), catalyzes the transformation of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP). For crystallographic studies, DXR from the human malaria parasite P. falciparum (PfDXR) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method in the presence of NADPH. X-ray diffraction data to 1.85 A resolution were collected from a monoclinic crystal form belonging to space group C2 with unit-cell parameters a = 168.89, b = 59.65, c = 86.58 A, beta = 117.8 degrees. Structural analysis by molecular replacement is in progress.

Crystallization and preliminary X-ray crystallographic studies of an exo-beta-D-glucosaminidase from Trichoderma reesei.

Chitosan is degraded to glucosamine (GlcN) by chitosanase and exo-beta-D-glucosaminidase (GlcNase). GlcNase from Trichoderma reesei (Gls93) is a 93 kDa extracellular protein composed of 892 amino acids. The enzyme liberates GlcN from the nonreducing end of the chitosan chain in an exo-type manner and belongs to glycoside hydrolase family 2. For crystallographic investigations, Gls93 was overexpressed in Pichia pastoris cells. The recombinant Gls93 had two molecular forms of approximately 105 kDa (Gls93-F1) and approximately 100 kDa (Gls93-F2), with the difference between them being caused by N-glycosylation. Both forms were crystallized by the hanging-drop vapour-diffusion method. Crystals of Gls93-F1 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 98.27, b = 98.42, c = 108.28 A, and diffracted to 1.8 A resolution. Crystals of Gls93-F2 belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.84, b = 81.62, c = 183.14 A, and diffracted to 2.4 A resolution. Both crystal forms were suitable for X-ray structure analysis at high resolution.

Crystallization of mouse S-adenosyl-L-homocysteine hydrolase.

S-adenosyl-L-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 A resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 A. Structural analysis by molecular replacement is in progress.

Crystallization and preliminary X-ray crystallographic study of phosphoglucose isomerase from Plasmodium falciparum.

Phosphoglucose isomerase (PGI) is a key enzyme in glycolysis and glycogenesis that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). For crystallographic studies, PGI from the human malaria parasite Plasmodium falciparum (PfPGI) was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 1.5 A resolution were collected from an orthorhombic crystal form belonging to space group P2(1)2(1)2(1) with unit-cell parameters a = 103.3, b = 104.1, c = 114.6 A. Structural analysis by molecular replacement is in progress.

Molecular basis for peroxisomal localization of tetrameric carbonyl reductase.

Pig heart peroxisomal carbonyl reductase (PerCR) belongs to the short-chain dehydrogenase/reductase family, and its sequence comprises a C-terminal SRL tripeptide, which is a variant of the type 1 peroxisomal targeting signal (PTS1) Ser-Lys-Leu. PerCR is imported into peroxisomes of HeLa cells when the cells are transfected with vectors expressing the enzyme. However, PerCR does not show specific targeting when introduced into the cells with a protein transfection reagent. To understand the structural basis for peroxisomal localization of PerCR, we determined the crystal structure of PerCR. Our data revealed that the C-terminal PTS1 of each subunit of PerCR was involved in intersubunit interactions and was buried in the interior of the tetrameric molecule. These findings indicate that the PTS1 receptor Pex5p in the cytosol recognizes the monomeric form of PerCR whose C-terminal PTS1 is exposed, and that this PerCR is targeted into the peroxisome, thereby forming a tetramer.

Molecular determinants for the stereospecific reduction of 3-ketosteroids and reactivity towards all-trans-retinal of a short-chain dehydrogenase/reductase (DHRS4).

DHRS4, a member of the short-chain dehydrogenase/reductase superfamily, reduces all-trans-retinal and xenobiotic carbonyl compounds. Human DHRS4 differs from other animal enzymes in kinetic constants for the substrates, particularly in its low reactivity to retinoids. We have found that pig, rabbit and dog DHRS4s reduce benzil and 3-ketosteroids into S-benzoin and 3alpha-hydroxysteroids, respectively, in contrast to the stereoselectivity of human DHRS4 which produces R-benzoin and 3beta-hydroxysteroids. Among substrate-binding residues predicted from the crystal structure of pig DHRS4, F158 and L161 in the animal DHRS4 are serine and phenylalanine, respectively, in the human enzyme. Double mutation (F158S/L161F) of pig DHRS4 led to an effective switch of its substrate affinity and stereochemistry into those similar to human DHRS4. The roles of the two residues in determining the stereospecificity in 3-ketosteroid reduction were confirmed by reverse mutation (S158F/F161L) in the human enzyme. The stereochemical control was evaluated by comparison of the 3D models of pig wild-type and mutant DHRS4s with the modeled substrates. Additional mutation of T177N into the human S158F/F161L mutant resulted in almost complete kinetic conversion into a pig DHRS4-type form, suggesting a role of N177 in forming the substrate-binding cavity through an intersubunit interaction in pig and other animal DHRS4s, and explaining why the human enzyme shows low reactivity towards retinoids.

Structural basis for recognition of cognate tRNA by tyrosyl-tRNA synthetase from three kingdoms.

The specific aminoacylation of tRNA by tyrosyl-tRNA synthetases (TyrRSs) relies on the identity determinants in the cognate tRNA(Tyr)s. We have determined the crystal structure of Saccharomyces cerevisiae TyrRS (SceTyrRS) complexed with a Tyr-AMP analog and the native tRNA(Tyr)(GPsiA). Structural information for TyrRS-tRNA(Tyr) complexes is now full-line for three kingdoms. Because the archaeal/eukaryotic TyrRSs-tRNA(Tyr)s pairs do not cross-react with their bacterial counterparts, the recognition modes of the identity determinants by the archaeal/eukaryotic TyrRSs were expected to be similar to each other but different from that by the bacterial TyrRSs. Interestingly, however, the tRNA(Tyr) recognition modes of SceTyrRS have both similarities and differences compared with those in the archaeal TyrRS: the recognition of the C1-G72 base pair by SceTyrRS is similar to that by the archaeal TyrRS, whereas the recognition of the A73 by SceTyrRS is different from that by the archaeal TyrRS but similar to that by the bacterial TyrRS. Thus, the lack of cross-reactivity between archaeal/eukaryotic and bacterial TyrRS-tRNA(Tyr) pairs most probably lies in the different sequence of the last base pair of the acceptor stem (C1-G72 vs G1-C72) of tRNA(Tyr). On the other hand, the recognition mode of Tyr-AMP is conserved among the TyrRSs from the three kingdoms.

Fragment-based discovery of the first nonpeptidyl inhibitor of an S46 family peptidase.

Antimicrobial resistance is a global public threat and raises the need for development of new antibiotics with a novel mode of action. The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to a new class of serine peptidases, family S46. Because S46 peptidases are not found in mammals, these enzymes are attractive targets for novel antibiotics. However, potent and selective inhibitors of these peptidases have not been developed to date. In this study, a high-resolution crystal structure analysis of PgDPP11 using a space-grown crystal enabled us to identify the binding of citrate ion, which could be regarded as a lead fragment mimicking the binding of a substrate peptide with acidic amino acids, in the S1 subsite. The citrate-based pharmacophore was utilized for in silico inhibitor screening. The screening resulted in an active compound SH-5, the first nonpeptidyl inhibitor of S46 peptidases. SH-5 and a lipophilic analog of SH-5 showed a dose-dependent inhibitory effect against the growth of P. gingivalis. The binding mode of SH-5 was confirmed by crystal structure analysis. Thus, these compounds could be lead structures for the development of selective inhibitors of PgDPP11.