[1-(Anthracen-9-ylmeth-yl)-1,4,7,10-tetra-aza-cyclododeca-ne]chlorido-zinc(II) nitrate.

In the title salt, [ZnCl(C23H30N4)]NO3, the central ZnII atom of the complex cation is coordinated in a square-pyramidal arrangement by four nitro-gen atoms from cyclen (1,4,7,10-tetra-aza-cyclo-dodeca-ne) in the basal plane and one chlorido ligand in the apical position. The anthracene group attached to cyclen contributes to the crystal packing through inter-molecular T-shaped π inter-actions. Additionally, the nitrate anion participates in inter-molecular N-H⋯O hydrogen bonds with cyclen.

(5-Fluoro-2,6-dioxo-1,2,3,6-tetra-hydro-pyrimidin-1-ido-κN 1)(1,4,8,11-tetra-aza-cyclo-tetra-decane-κ4 N)zinc(II) perchlorate.

In the structure of the title complex, [Zn(C4H2FN2O2)(C10H24N4)]ClO4, the zinc(II) ion forms coordination bonds with the four nitro-gen atoms of cyclam (1,4,8,11-tetra-aza-cyclo-tetra-decane or [14]aneN4) as well as with the nitro-gen atom of a deprotonated 5-fluoro-uracil ion (FU-). Cyclam adopts a trans-I type conformation within this structure. The coordination structure of the zinc(II) ion is a square pyramid with a distorted base plane formed by the four nitro-gen atoms of the cyclam. FU- engages in inter-molecular hydrogen bonding with neighboring FU- mol-ecules and with the cyclam mol-ecule.

Hydroxyhexylitaconic acids as potent IMP-type metallo-ß-lactamase inhibitors for controlling carbapenem resistance in Enterobacterales.

Metallo-ß-lactamases (MBLs) represent one of the main causes of carbapenem resistance in the order Enterobacterales. To combat MBL-producing carbapenem-resistant Enterobacterales, the development of MBL inhibitors can restore carbapenem efficacy for such resistant bacteria. Microbial natural products are a promising source of attractive seed compounds for the development of antimicrobial agents. Here, we report that hydroxyhexylitaconic acids (HHIAs) produced by a member of the genus Aspergillus can suppress carbapenem resistance conferred by MBLs, particularly IMP (imipenemase)-type MBLs. HHIAs were found to be competitive inhibitors with micromolar orders of magnitude against IMP-1 and showed weak inhibitory activity toward VIM-2, while no inhibitory activity against NDM-1 was observed despite the high dosage. The elongated methylene chains of HHIAs seem to play a crucial role in exerting inhibitory activity because itaconic acid, a structural analog without long methylene chains, did not show inhibitory activity against IMP-1. The addition of HHIAs restored meropenem and imipenem efficacy to satisfactory clinical levels against IMP-type MBL-producing Escherichia coli and Klebsiella pneumoniae clinical isolates. Unlike EDTA and Aspergillomarasmine A, HHIAs did not cause the loss of zinc ions from the active site, resulting in the structural instability of MBLs. X-ray crystallography and in silico docking simulation analyses revealed that two neighboring carboxylates of HHIAs coordinated with two zinc ions in the active sites of VIM-2 and IMP-1, which formed a key interaction observed in MBL inhibitors. Our results indicated that HHIAs are promising for initiating the design of potent inhibitors of IMP-type MBLs.IMPORTANCEThe number and type of metallo-ß-lactamase (MΒL) are increasing over time. Carbapenem resistance conferred by MΒL is a significant threat to our antibiotic regimen, and the development of MΒL inhibitors is urgently required to restore carbapenem efficacy. Microbial natural products have served as important sources for developing antimicrobial agents targeting pathogenic bacteria since the discovery of antibiotics in the mid-20th century. MΒL inhibitors derived from microbial natural products are still rare compared to those derived from chemical compound libraries. Hydroxyhexylitaconic acids (HHIAs) produced by members of the genus Aspergillus have potent inhibitory activity against clinically relevant IMP-type MBL. HHIAs may be good lead compounds for the development of MBL inhibitors applicable for controlling carbapenem resistance in IMP-type MBL-producing Enterobacterales.

Clinical isolates of Streptococcus mitis/oralis-related species with reduced carbapenem susceptibility, harboring amino acid substitutions in penicillin-binding proteins in Japan.

Streptococcus mitis/oralis group isolates with reduced carbapenem susceptibility have been reported, but its isolation rate in Japan is unknown. We collected 356 clinical α-hemolytic streptococcal isolates and identified 142 of them as S. mitis/oralis using partial sodA sequencing. The rate of meropenem non-susceptibility was 17.6% (25/142). All 25 carbapenem-non-susceptible isolates harbored amino acid substitutions in/near the conserved motifs in PBP1A, PBP2B, and PBP2X. Carbapenem non-susceptibility is common among S. mitis/oralis group isolates in Japan.

ß-Lactam Susceptibility of Streptococcus dysgalactiae subsp. equisimilis.

All clinical isolates of Streptococcus dysgalactiae subsp. equisimilis (SDSE) are considered susceptible to ß-lactams, the first-line drugs used to treat SDSE infections. However, given that penicillin-non-susceptible SDSE strains have been isolated in Denmark, in this study, we aimed to identify ß-lactam-non-susceptible clinical isolates of SDSE in Japan. In 2018, we collected 150 clinical isolates of S. dysgalactiae, and species identification was performed using a Rapid ID Strep API kit. The minimum inhibitory concentrations (MIC) of six ß-lactams (penicillin G, oxacillin, ceftizoxime, ceftibuten, cefoxitin, and cefaclor) were determined for the 85 clinical isolates identified as SDSE using the agar dilution method standardized by the Clinical & Laboratory Standards Institute. The MIC ranges of penicillin G, oxacillin, ceftizoxime, ceftibuten, cefoxitin, and cefaclor were 0.007-0.06, 0.03-0.12, 0.015-0.06, 0.25-2, 0.12-2, and 0.06-0.5 µg/mL, respectively. None of the clinical isolates of SDSE were non-susceptible to penicillin G, indicating that all 85 clinical isolates of SDSE were susceptible to ß-lactams. Our findings indicate that almost all clinical isolates of SDSE, from several prefectures of Japan, are still susceptible to ß-lactams. Nevertheless, there remains a need for continuous and careful monitoring of drug susceptibility among clinical SDSE isolates in Japan.

Aqua-{µ-1,4-bis-[(1,4,7,10-tetra-aza-cyclo-dodecan-1-yl)meth-yl]benzene}(nitrato-κO)dicopper(II) tris-(nitrate) trihydrate.

In the title dinuclear CuII complex, [Cu2(NO3)(C24H46N8)(H2O)](NO3)3·3H2O, the two CuII mol-ecules both have a square-pyramidal geometry, but the ligands in the axial positions are different: a water mol-ecule and a nitrate ion. All nitrate ions, water mol-ecules, and N-H groups are involved in an inter-molecular hydrogen-bond network.

Structural Characterization of Zinc(II)/Cobalt(II) Complexes of Chiral N-(Anthracen-9-yl)methyl-N,N-bis(2-picolyl)amine and Evaluation of DNA Photocleavage Activity.

We designed and synthesized a chiral ligand N-(anthracen-9-ylmethyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)ethanamine (APPE) DNA photocleavage agent to investigate the effects of chirality of bis(2-picolyl)amine on the DNA photocleavage activity of metal complexes. The structures of ZnII and CoII complexes in APPE were analyzed via X-ray crystallography and fluorometric titration. APPE formed metal complexes with a 1 : 1 stoichiometry in both the crystalline and solution states. Fluorometric titration was used to show that the ZnII and CoII association constants of these complexes (log Kas) were 4.95 and 5.39, respectively. The synthesized complexes were found to cleave pUC19 plasmid DNA when irradiated at 370 nm. The DNA photocleavage activity of the ZnII complex was higher than that of the CoII complex. The absolute configuration of the methyl-attached carbon did not affect DNA cleavage activity and, unfortunately, an achiral APPE derivative without the methyl group (ABPM) was found to perform DNA photocleavage more effectively than APPE. One reason for this may be that the methyl group suppressed the structural flexibility of the photosensitizer. These results will be useful for the design of new photoreactive reagents.

Improved disk diffusion method for simple detection of group B streptococci with reduced penicillin susceptibility (PRGBS).

We used 73 group B Streptococcus with reduced penicillin susceptibility (PRGBS) isolates and determined more rational cutoff values of previously developed disk diffusion method for detecting PRGBS using oxacillin, ceftizoxime, and ceftibuten disks. Using the novel cutoff values, the three disks showed high sensitivity and specificity, which were above 90.0%.

Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-ß-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution.

Gram-negative bacteria producing metallo-ß-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the ß-lactam ring present in a broad range of antibiotics, such as N-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (kfast = 1.76 h-1, kslow = 0.108 h-1 for IMP-1, and kfast = 14.0 h-1 and kslow = 1.66 h-1 for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against ß-lactam antibiotics, and the inhibitory effect of thiol compounds.

Bis(nitrato-κO)(1,4,8,11-tetra-aza-cyclo-tetra-decane-κ4 N)zinc(II) methanol monosolvate.

The two ZnII atoms in the crystal structure of the title complex, [Zn(NO3)2(C10H24N4)]·CH3OH, have a distorted octa-hedral coordination sphere, defined by 1,4,8,11-tetra-aza-cyclo-tetra-decane (cyclam) N atoms in the equatorial plane and nitrate O atoms in the axial sites. The conformation of the cyclam is trans-III (R, R, S, S), which is typical for metal-cyclam complexes. Nitrate anions are involved in intra- and inter-molecular hydrogen bonding with the N-H groups of the ZnII-cyclam unit. Together with the methanol solvent mol-ecule, the hydrogen-bonding network connects the ZnII-cyclam units into ribbons running parallel to the a axis.

A Rapid and Sensitive Determination of Histamine in Mast Cells Using a Didansyl Derivatization Method.

BACKGROUND: Mast cells play a central role in allergic responses such as food allergy, asthma, allergic rhinitis, and allergic conjunctivitis. Symptoms in the early phase of these allergic diseases are primarily caused by histamine. However, due to the high histidine content in the cytosol and low histamine content in secretory granules, separating and quantifying histamine from histidine is often difficult. OBJECTIVES: We studied a method for rapid and sensitive quantitation of mast cell-derived histamine and evaluated its application to allergic disease research. METHODS: Bone marrow-derived mouse mast cells (BMMCs) were employed in this study. IgE-sensitized BMMCs were activated by FcεRI cross-linking. After activation, both the histamine released to the supernatant and histamine remaining in BMMCs were didansylated and then analyzed by high-performance liquid chromatography with fluorescence detection (HPLC-FD). Didansyl histamine was synthesized as a standard material. RESULTS: Synthetic didansyl histamine was detected by HPLC-FD with a peak retention time of 18.5 min. Very high linearity of the standard curve was maintained at concentrations of 10 pg/µL or less when the didansyl histamine method was used. This method enables detection of histamine released from 1 × 105 BMMCs. In addition, the histamine concentration in the supernatant due to spontaneous release was also determined. Finally, the ratio of histamine release was highly correlated with the degranulation ratio. CONCLUSION: These results indicate that the proposed method using didansylated histamine to determine mast cell-derived histamine is highly useful for allergy research applications.

Molecular Epidemiology of Enterobacter cloacae Complex Isolates with Reduced Carbapenem Susceptibility Recovered by Blood Culture.

The Enterobacter cloacae complex (ECC) is one of the most common causes of bacteremia and leads to poor clinical outcomes. The aim of this study was to clarify the antimicrobial susceptibility profiles and genetic backgrounds of non-carbapenemase-producing reduced-carbapenem-susceptible (RCS) ECC blood isolates in Japan using agar dilution antimicrobial susceptibility testing, whole-genome sequencing, and quantitative polymerase chain reaction for ampC, ompC, and ompF transcripts. Forty-two ECC blood isolates were categorized into RCS and carbapenem-susceptible groups based on the minimum inhibitory concentration of imipenem. The RCS ECC blood isolates belonged to distinct species and sequence types and produced varying class C ß-lactamases. The E. roggenkampii, E. asburiae, and E. bugandensis isolates belonged only to the RCS group. Some E. hormaechei ssp. steigerwaltii isolates from the RCS group exhibited AmpC overexpression caused by amino acid substitutions in AmpD and AmpR along with ompF downregulation. These findings suggest that non-carbapenemase-producing RCS ECC blood isolates are genetically diverse.

Isolation of Group A Streptococci with Reduced In Vitro ß-Lactam Susceptibility Harboring Amino Acid Substitutions in Penicillin-Binding Proteins in Japan.

Streptococcus pyogenes (group A Streptococcus [GAS]) has long been regarded as being susceptible to ß-lactams. However, amino acid substitutions in penicillin-binding protein 2X (PBP2X) conferring reduced in vitro ß-lactam susceptibility have been indicated since 2019 in the United States and Iceland. Here, we report the first isolation of Streptococcus pyogenes possessing the PBP2X substitution conferring reduced in vitro ß-lactam susceptibility in Asia; however, the MICs were below the susceptible breakpoint of the CLSI.

Practical Agar-Based Disk Diffusion Tests Using Sulfamoyl Heteroarylcarboxylic Acids for Identification of Subclass B1 Metallo-ß-Lactamase-Producing Enterobacterales.

The worldwide distribution of carbapenemase-producing Enterobacterales (CPE) is a serious public health concern as they exhibit carbapenem resistance, thus limiting the choice of antimicrobials for treating CPE infections. Combination treatment with a ß-lactam and one of the newly approved ß-lactamase inhibitors, such as avibactam, relebactam, or vaborbactam, provides a valuable tool to cope with CPE; however, these inhibitors are active only against serine-type carbapenemases and not against metallo-ß-lactamases (MßLs). Therefore, it is important to readily differentiate carbapenemases produced by CPE by using simple and reliable methods in order to choose an appropriate treatment. Here, we developed three practical agar-based disk diffusion tests (double-disk synergy test [DDST], disk potentiation test, and modified carbapenem inactivation method [mCIM]) to discriminate the production of subclass B1 MßLs, such as IMP-, NDM-, and VIM-type MßLs, from the other carbapenemases, especially serine-type carbapenemases. This was accomplished using B1 MßL-specific sulfamoyl heteroarylcarboxylic acid inhibitors, 2,5-dimethyl-4-sulfamoylfuran-3-carboxylic acid (SFC) and 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), originally developed by us. The DDST and mCIM using SFC and SPC revealed high sensitivity (95.3%) and specificity (100%) in detecting B1 MßL-producing Enterobacterales. In the disk potentiation test, the sensitivities using SFC and SPC were 89.1% and 93.8%, respectively, whereas the specificities for both were 100%. These methods are simple and inexpensive and have a high accuracy rate. These methods would therefore be of immense assistance in the specific detection and discrimination of B1 MßL-producing Enterobacterales in clinical microbiology laboratories and would lead to better prevention against infection with such multidrug-resistant bacteria in clinical settings.

Crystal Structures of Metallo-ß-Lactamase (IMP-1) and Its D120E Mutant in Complexes with Citrate and the Inhibitory Effect of the Benzyl Group in Citrate Monobenzyl Ester.

The emergence and rapid spread of carbapenem-resistant pathogens producing metallo-ß-lactamases such as IMP-1 and NDM-1 have been of great concern in the global clinical setting. The X-ray crystal structures of IMP-1 from Serratia marcescens and its single mutant, D120E, in complexes with citrate were determined at resolutions of 2.00 and 1.85 Å, respectively. Two crystal structures indicate that a single mutation at position 120 caused a structural change around Zn1, where the geometry changes from a tetrahedron in the native IMP-1 to a square pyramid in D120E. Based on these two complex structures, the authors synthesized citrate monobenzyl ester 1 to evaluate the structural requirement for the inhibitory activity against IMP-1 and compared the inhibitory activities with nonsubstituted citrate. The introduction of a benzyl group into citrate enhanced the inhibitory activity in comparison to citrate (IC50 > 5 mM).

DNA-cleavage activity of the iron(II) complex with optically active ligands, meta- and para-xylyl-linked N',N'-dipyridylmethyl-cyclohexane-1,2-diamine.

DNA-cleavage agents such as bleomycin have potential anticancer applications. The development of a DNA-cleavage reagent that recognizes specific sequences allows the development of cancer therapy with reduced side effects. In this study, to develop novel compounds with specific DNA-cleavage activities, we synthesized optically active binuclear ligands, (1R,1'R,2R,2'R)-N1,N1'-(meta/para-phenylenebis(methylene))bis(N2,N2-bis(pyridin-2-ylmethyl)cyclohexane-1,2-diamine) and their enantiomers. The DNA-cleavage activities of these compounds were investigated in the presence of Fe(II)SO4 and sodium ascorbate. The obtained results indicated that the Fe(II) complexes of those compounds efficiently cleave DNA and that their cleavage was subtle sequence-selective. Therefore, we succeeded in developing compounds that can be used as small-molecule drugs for cancer chemotherapy.

Design and synthesis of an anthranyl bridged optically active dinuclear iron(II)-ligand and evaluation of DNA-cleaving activity.

It is necessary to design a ligand that is compatible with the target molecule to optimally use the DNA-cleaving ability of metal complexes. In this study, we synthesized an optically active dinuclear ligand, (1R,1'R,2R,2'R)-N1,N1'-(anthracene-1,8-diylbis(methylene))bis(N2,N2-bis(pyridin-2-ylmethyl)cyclohexane-1,2-diamine) (R-ABDC, 4a) and its enantiomer (S-ABDC, 4b). We then prepared their Fe(II) complexes by mixing the ligand with FeSO4·7H2O in situ and investigated DNA-cleaving activities using plasmid DNA in the presence of excess sodium ascorbate at atmospheric conditions. The Fe(II) complexes efficiently cleaved DNA and selectively recognized two consecutive A and/or T sequences.

Daptomycin Susceptibility of Group B Streptococcus.

We previously reported the emergence and high prevalence of group B streptococci (GBS) with reduced penicillin susceptibility (PRGBS) clinical isolates in Japan. PRGBS tend to be non-susceptible to macrolides and fluoroquinolones. In our previous study, we found that the minimum inhibitory concentration (MIC) of daptomycin for one clinical isolate of GBS was above the susceptible breakpoint settled by the Clinical and Laboratory Standards Institute (CLSI). This suggests the possibility of the unrecognized spread of daptomycin-non-susceptible clinical GBS isolates in Japan. This study aimed to analyze the daptomycin susceptibility in 1,046 clinical GBS isolates that were recovered after the approval of daptomycin in Japan. MICs of daptomycin for the 1,046 clinical isolates were determined by the microdilution method recommended by the CLSI. The MIC range was 0.12-1 µg/mL, and the MIC50 and MIC90 were 0.5 µg/mL and 1 µg/mL, respectively. All the GBS isolates evaluated in this study were susceptible to daptomycin. Therefore, at present, daptomycin might be considered as a new option to treat GBS infections, especially multidrug-resistant PRGBS infections.

Sulfamoyl Heteroarylcarboxylic Acids as Promising Metallo-ß-Lactamase Inhibitors for Controlling Bacterial Carbapenem Resistance.

Production of metallo-ß-lactamases (MBLs), which hydrolyze carbapenems, is a cause of carbapenem resistance in Enterobacteriaceae Development of effective inhibitors for MBLs is one approach to restore carbapenem efficacy in carbapenem-resistant Enterobacteriaceae (CRE). We report here that sulfamoyl heteroarylcarboxylic acids (SHCs) can competitively inhibit the globally spreading and clinically relevant MBLs (i.e., IMP-, NDM-, and VIM-type MBLs) at nanomolar to micromolar orders of magnitude. Addition of SHCs restored meropenem efficacy against 17/19 IMP-type and 7/14 NDM-type MBL-producing Enterobacteriaceae to satisfactory clinical levels. SHCs were also effective against IMP-type MBL-producing Acinetobacter spp. and engineered Escherichia coli strains overproducing individual minor MBLs (i.e., TMB-2, SPM-1, DIM-1, SIM-1, and KHM-1). However, SHCs were less effective against MBL-producing Pseudomonas aeruginosa Combination therapy with meropenem and SHCs successfully cured mice infected with IMP-1-producing E. coli and dually NDM-1/VIM-1-producing Klebsiella pneumoniae clinical isolates. X-ray crystallographic analyses revealed the inhibition mode of SHCs against MBLs; the sulfamoyl group of SHCs coordinated to two zinc ions, and the carboxylate group coordinated to one zinc ion and bound to positively charged amino acids Lys224/Arg228 conserved in MBLs. Preclinical testing revealed that the SHCs showed low toxicity in cell lines and mice and high stability in human liver microsomes. Our results indicate that SHCs are promising lead compounds for inhibitors of MBLs to combat MBL-producing CRE.IMPORTANCE Carbapenem antibiotics are the last resort for control of severe infectious diseases, bloodstream infections, and pneumonia caused by Gram-negative bacteria, including Enterobacteriaceae However, carbapenem-resistant Enterobacteriaceae (CRE) strains have spread globally and are a critical concern in clinical settings because CRE infections are recognized as a leading cause of increased mortality among hospitalized patients. Most CRE produce certain kinds of serine carbapenemases (e.g., KPC- and GES-type ß-lactamases) or metallo-ß-lactamases (MBLs), which can hydrolyze carbapenems. Although effective MBL inhibitors are expected to restore carbapenem efficacy against MBL-producing CRE, no MBL inhibitor is currently clinically available. Here, we synthesized 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), which is a potent inhibitor of MBLs. SPC is a remarkable lead compound for clinically useful MBL inhibitors and can potentially provide a considerable benefit to patients receiving treatment for lethal infectious diseases caused by MBL-producing CRE.

4-Amino-2-Sulfanylbenzoic Acid as a Potent Subclass B3 Metallo-ß-Lactamase-Specific Inhibitor Applicable for Distinguishing Metallo-ß-Lactamase Subclasses.

The number of cases of infection with carbapenem-resistant Enterobacteriaceae (CRE) has been increasing and has become a major clinical and public health concern. Production of metallo-ß-lactamases (MBLs) is one of the principal carbapenem resistance mechanisms in CRE. Therefore, developing MBL inhibitors is a promising strategy to overcome the problems of carbapenem resistance conferred by MBLs. To date, the development and evaluation of MBL inhibitors have focused on subclass B1 MBLs but not on B3 MBLs. In the present study, we searched for B3 MBL (specifically, SMB-1) inhibitors and found thiosalicylic acid (TSA) to be a potent inhibitor of B3 SMB-1 MBL (50% inhibitory concentration [IC50], 0.95 µM). TSA inhibited the purified SMB-1 to a considerable degree but was not active against Escherichia coli cells producing SMB-1, as the meropenem (MEM) MIC for the SMB-1 producer was only slightly reduced with TSA. We then introduced a primary amine to TSA and synthesized 4-amino-2-sulfanylbenzoic acid (ASB), which substantially reduced the MEM MICs for SMB-1 producers. X-ray crystallographic analyses revealed that ASB binds to the two zinc ions, Ser221, and Thr223 at the active site of SMB-1. These are ubiquitously conserved residues across clinically relevant B3 MBLs. ASB also significantly inhibited other B3 MBLs, including AIM-1, LMB-1, and L1. Therefore, the characterization of ASB provides a starting point for the development of optimum B3 MBL inhibitors.