Benzimidazole and Benzoxazole Zinc Chelators as Inhibitors of Metallo-β-Lactamase NDM-1

Bacterial expression of β-lactamases, which hydrolyze β-lactam antibiotics, contributes to the growing threat of antibacterial drug resistance. Metallo-β-lactamases, such as NDM-1, use catalytic zinc ions in their active sites and hydrolyze nearly all clinically available β-lactam antibiotics. Inhibitors of metallo-β-lactamases are urgently needed to overcome this resistance mechanism. Zinc-binding compounds are promising leads for inhibitor development, as many NDM-1 inhibitors contain zinc-binding pharmacophores. Here, we evaluated 13 chelating agents containing benzimidazole and benzoxazole scaffolds as NDM-1 inhibitors. Six of the compounds showed potent inhibitory activity with IC₅₀ values as low as 0.38 μM, and several compounds restored the meropenem susceptibility of NDM-1-expressing E. coli. Spectroscopic and docking studies suggest ternary complex formation as the mechanism of inhibition, making these compounds promising for development as NDM-1 inhibitors.     

Mechanistic Investigations of Metallo-β-lactamase Inhibitors: Strong Zinc Binding Is Not Required for Potent Enzyme Inhibition**

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial enzymes that inactivate essentially all classes of β-lactam antibiotics including last-resort carbapenems. At present there are no clinically approved MBL inhibitors, and in order to develop such agents it is essential to understand their inhibitory mechanisms. Herein, we describe a comprehensive mechanistic study of a panel of structurally distinct MBL inhibitors reported in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration (IC₅₀) for each inhibitor against MBLs belonging to the NDM and IMP families. In addition, the binding affinities of the inhibitors for Zn²⁺, Ca²⁺ and Mg²⁺ were assessed by using isothermal titration calorimetry (ITC). We also compared the ability of the different inhibitors to resensitize a highly resistant MBL-expressing Escherichia coli strain to meropenem. These investigations reveal clear differences between the MBL inhibitors studied in terms of their IC₅₀ value, metal binding ability, and capacity to synergize with meropenem. Notably, our studies demonstrate that potent MBL inhibition and synergy with meropenem are not explicitly dependent on the capacity of an inhibitor to strongly chelate zinc.     

Modulation of Aβ42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors

The aggregation of amyloid-β 42 (Aβ42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aβ42 aggregation kinetics, characterized by the quantification of Aβ42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aβ42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.     

Investigation of Dipicolinic Acid Isosteres for the Inhibition of Metallo-β-Lactamases

New Delhi metallo-β-lactamase-1 (NDM-1) poses an immediate threat to our most effective and widely prescribed drugs, the β-lactam-containing class of antibiotics. There are no clinically relevant inhibitors to combat NDM-1, despite significant efforts toward their development. Inhibitors that use a carboxylic acid motif for binding the Zn^II ions in the active site of NDM-1 make up a large portion of the >500 inhibitors reported to date. New and structurally diverse scaffolds for inhibitor development are needed urgently. Herein we report the isosteric replacement of one carboxylate group of dipicolinic acid (DPA) to obtain DPA isosteres with good inhibitory activity against NDM-1 (and related metallo-β-lactamases, IMP-1 and VIM-2). It was determined that the choice of carboxylate isostere influences both the potency of NDM-1 inhibition and the mechanism of action. Additionally, we show that an isostere with a metal-stripping mechanism can be re-engineered into an inhibitor that favors ternary complex formation. This work provides a roadmap for future isosteric replacement of routinely used metal binding motifs (i.e., carboxylic acids) for the generation of new entities in NDM-1 inhibitor design and development.     

β-Actin Peptide-Based Inhibitors of Histidine Methyltransferase SETD3

SETD3 was recently identified as the histidine methyltransferase responsible for N³-methylation of His73 of β-actin in humans. Overexpression of SETD3 is associated with several diseases, including breast cancer. Here, we report a development of actin-based peptidomimetics as inhibitors of recombinantly expressed human SETD3. Substitution of His73 by simple natural and unnatural amino acids led to selected β-actin peptides with high potency against SETD3 in MALDI-TOF MS assays. The selenomethionine-containing β-actin peptide was found to be the most potent SETD3 inhibitor (IC₅₀=161 nM). Supporting our inhibition assays, a combination of computational docking and molecular dynamics simulations revealed that the His73 binding pocket for β-actin in SETD3 is rigid and accommodates the inhibitor peptides with similar binding modes. Collectively, our work demonstrates that actin-based peptidomimetics can act as potent SETD3 inhibitors and provide a basis for further development of highly potent and selective inhibitors of SETD3.     

1,2,4-Triazole-3-Thione Analogues with a 2-Ethylbenzoic Acid at Position 4 as VIM-type Metallo-β-Lactamase Inhibitors

Metallo-β-lactamases (MBLs) are increasingly involved as a major mechanism of resistance to carbapenems in relevant opportunistic Gram-negative pathogens. Unfortunately, clinically efficient MBL inhibitors still represent an unmet medical need. We previously reported several series of compounds based on the 1,2,4-triazole-3-thione scaffold. In particular, Schiff bases formed between diversely 5-substituted-4-amino compounds and 2-carboxybenzaldehyde were broad-spectrum inhibitors of VIM-type, NDM-1 and IMP-1 MBLs. Unfortunately, these compounds were unable to restore antibiotic susceptibility of MBL-producing bacteria, probably because of poor penetration and/or susceptibility to hydrolysis. To improve their microbiological activity, we synthesized and characterized compounds where the hydrazone-like bond of the Schiff base analogues was replaced by a stable ethyl link. This small change resulted in a narrower inhibition spectrum, as all compounds were poorly or not inhibiting NDM-1 and IMP-1, but showed a significantly better activity on VIM-type enzymes, with K_i values in the μM to sub-μM range. The resolution of the crystallographic structure of VIM-2 in complex with one of the best inhibitors yielded valuable information about their binding mode. Interestingly, several compounds were shown to restore the β-lactam susceptibility of VIM-type-producing E. coli laboratory strains and also of K. pneumoniae clinical isolates. In addition, selected compounds were found to be devoid of toxicity toward human cancer cells at high concentration, thus showing promising safety.     

Discovery and Mechanism of Type III Secretion System Inhibitors

The Type III Secretion System (TTSS) is indispensable for virulence of many Gram-negative pathogenic bacteria, including Escherichia coli, Salmonella spp., Yersinia spp., Vibrio spp., Chlamydia spp., Shigella spp., Pseudomonas spp., Xanthomonas spp., and Auromonas spp. Such pathogenic bacteria are responsible for diseases such as plague, shigellosis, chlamydia, cholera, pneumonia, and gastroenteritis. This review offers insights into the known inhibitors of the TTSS, their discovery, and their mode of action.     

Synthesis of 1′-Aryl-1,3′-bi-β-carbolines and Their Saturated Counterparts

1-Aryl-β-carboline-3-carbaldehyde building blocks have been used as the starting materials for the synthesis of novel bis-β-carboline derivatives. In the two-step synthesis the Pictet–Spengler reaction was used before the dehydrogenation of the polycyclic compound to the aromatic derivatives.     

Synthesis of Novel Pyridine-Carboxylates as Small-Molecule Inhibitors of Human Aspartate/Asparagine-β-Hydroxylase

The human 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) is a potential medicinal chemistry target for anticancer therapy. AspH is present on the cell surface of invasive cancer cells and accepts epidermal growth factor-like domain (EGFD) substrates with a noncanonical (i. e., Cys 1–2, 3–4, 5–6) disulfide pattern. We report a concise synthesis of C-3-substituted derivatives of pyridine-2,4-dicarboxylic acid (2,4-PDCA) as 2OG competitors for use in SAR studies on AspH inhibition. AspH inhibition was assayed by using a mass spectrometry-based assay with a stable thioether analogue of a natural EGFD AspH substrate. Certain C-3-substituted 2,4-PDCA derivatives were potent AspH inhibitors, manifesting selectivity over some, but not all, other tested human 2OG oxygenases. The results raise questions about the use of pyridine-carboxylate-related 2OG analogues as selective functional probes for specific 2OG oxygenases, and should aid in the development of AspH inhibitors suitable for in vivo use.     

The Design of Potent,Selective and Drug-Like RGD αvβ1 Small-Molecule Inhibitors Derived from non-RGD α4β1 Antagonists

Up to 45 % of deaths in developed nations can be attributed to chronic fibroproliferative diseases, highlighting the need for effective therapies. The RGD (Arg-Gly-Asp) integrin αvβ1 was recently investigated for its role in fibrotic disease, and thus warrants therapeutic targeting. Herein we describe the identification of non-RGD hit small-molecule αvβ1 inhibitors. We show that αvβ1 activity is embedded in a range of published α4β1 (VLA-4) ligands; we also demonstrate how a non-RGD integrin inhibitor (of α4β1 in this case) was converted into a potent non-zwitterionic RGD integrin inhibitor (of αvβ1 in this case). We designed urea ligands with excellent selectivity over α4β1 and the other αv integrins (αvβ3, αvβ5, αvβ6, αvβ8). In silico docking models and density functional theory (DFT) calculations aided the discovery of the lead urea series.     

An Angle on MK2 Inhibition—Optimization and Evaluation of Prevention of Activation Inhibitors

The mitogen-activated protein kinase p38α pathway has been an attractive target for the treatment of inflammatory conditions such as rheumatoid arthritis. While a number of p38α inhibitors have been taken to the clinic, they have been limited by their efficacy and toxicological profile. A lead identification program was initiated to selectively target prevention of activation (PoA) of mitogen-activated protein kinase-activated protein kinase 2 (MK2) rather than mitogen- and stress-activated protein kinase 1 (MSK1), both immediate downstream substrates of p38α, to improve the efficacy/safety profile over direct p38α inhibition. Starting with a series of pyrazole amide PoA MK2 inhibitor leads, and guided by structural chemistry and rational design, a highly selective imidazole 9 (2-(3′-(2-amino-2-oxoethyl)-[1,1′-biphenyl]-3-yl)-N-(5-(N,N-dimethylsulfamoyl)-2-methylphenyl)-1-propyl-1H-imidazole-5-carboxamide) and the orally bioavailable imidazole 18 (3-methyl-N-(2-methyl-5-sulfamoylphenyl)-2-(o-tolyl)imidazole-4-carboxamide) were discovered. The PoA concept was further evaluated by protein immunoblotting, which showed that the optimized PoA MK2 compounds, despite their biochemical selectivity against MSK1 phosphorylation, behaved similarly to p38 inhibitors in cellular signaling. This study highlights the importance of selective tool compounds in untangling complex signaling pathways, and although 9 and 18 were not differentiated from p38α inhibitors in a cellular context, they are still useful tools for further research directed to understand the role of MK2 in the p38α signaling pathway.     

Design and Efficient Synthesis of RalA Inhibitors Containing the Dihydro-α-carboline Scaffold

Ras-related protein RalA is a member of the Ras small GTPases superfamily. Its activation plays an important role in regulating tumor initiation, invasion, migration, and metastasis. In this study, we designed a new type of RalA inhibitor containing a dihydro-α-carboline scaffold. The structurally new dihydro-α-carboline derivatives could be efficiently synthesized in good yields through a newly developed three-component [3+2+1] cyclization reaction. Evaluation of the biological activity showed that some of the dihydro-α-carboline derivatives can inhibit RalA/B and proliferative activities of NSCLC cell lines. The 4-(pyridin-3-yl)-dihydro-α-carboline compound ( 3 o ) was found to be the most potent derivative, with IC₅₀ values of 0.43±0.03, 0.64±0.07, 0.93±0.10, and 1.54±0.15 μM against A549, H1299, H460, and H1975 cells, respectively. Mechanism investigation suggested that 3 o inhibits the RalA/B activation of A549, down-regulates Bcl-2, stimulates cytochrome c and PARP cleavage, and induces cell apoptosis. A molecular docking study revealed that 3 o can form stable hydrogen bonds with residues of RalA. Moreover, amide-π and alkyl-π interactions also contributed to the affinity between 3 o and RalA.     

Identification of Specific and Nonspecific Inhibitors of Bacillus anthracis Type III Pantothenate Kinase (PanK)

Antibiotics with novel mechanisms of action are desperately needed to combat the increasing rates of multidrug-resistant infections. Bacterial pantothenate kinase (PanK) has emerged as a target of interest to cut off the biosynthesis of coenzyme A. Herein we report the results of an in vitro high-throughput screen of over 10 000 small molecules against Bacillus anthracis PanK, as well as a follow-up screen of hits against PanK isolated from Pseudomonas aeruginosa and Burkholderia cenocepacia. Nine hits are structurally categorized and analyzed to set the stage for future drug development.     

Structure,Function and Mechanism of N-Glycan Processing Enzymes: endo-α-1,2-Mannanase and endo-α-1,2-Mannosidase

While most glycosidases that act on N-linked glycans remove a single sugar residue at a time, endo-α-1,2-mannosidases and endo-α-1,2-mannanases of glycoside hydrolase family GH99 cut within a chain and remove two or more sugar residues. They are stereochemically retaining enzymes that use an enzymatic mechanism involving an epoxide intermediate. Human endo-α-1,2-mannosidase (MANEA) trims glucosylated mannose residues; the endomannosidase pathway provides a glucosidase-independent pathway for glycoprotein maturation. Cell-active MANEA inhibitors alter N-glycan processing and reduce infectivity of dengue virus, demonstrating that MANEA has potential as a host-directed antiviral target. Sequence-related enzymes from gut Bacteroides spp. exhibit endo-α-1,2-mannosidase activity and are a fruitful test bed for structure-guided inhibitor development. The genes encoding the Bacteroides spp. enzymes sit within polysaccharide utilization loci and are preferential endo-α-1,2-mannanases.     

Design,Synthesis and Discovery of N,N’-Carbazoyl-aryl-urea Inhibitors of Zika NS5 Methyltransferase and Virus Replication

The recent outbreaks of Zika virus (ZIKV) infection worldwide make the discovery of novel antivirals against flaviviruses a research priority. This work describes the identification of novel inhibitors of ZIKV through a structure-based virtual screening approach using the ZIKV NS5-MTase. A novel series of molecules with a carbazoyl-aryl-urea structure has been discovered and a library of analogues has been synthesized. The new compounds inhibit ZIKV MTase with IC₅₀ between 23–48 μM. In addition, carbazoyl-aryl-ureas also proved to inhibit ZIKV replication activity at micromolar concentration.     

Comparison of electrostatic collection and liquid impinging methods when collecting airborne house dust allergens,endotoxin and (1,3)-β-d-glucans

The use of electrostatic collection was investigated in quantifying airborne environmental allergens and toxins. The experiments were conducted with two 96-well plates filled with water and placed into the electrostatic sampler designed in this study. The combinations of different electrostatic fields: 0.63, 1.25 kV/cm, and different sampling flow rates: 5, 12.5 L/min, were tested with the electrostatic sampler. As a reference, a BioSampler operating at 12.5 L/min was simultaneously placed in the same environments. The sampling lasted for 40 min both for electrostatic sampler and the BioSampler in each test. House dust allergens, endotoxin and (1,3)-β-d-glucans in the air samples collected were analyzed using enzyme-linked immunosorbent assay (ELISA) and Limulus amebocyte lysate (LAL) method, respectively. The entire experiments were conducted both in office environment and hotel rooms.The concentrations of airborne allergens (Der p 1 and Der f 1) and toxins (endotoxin and (1,3)-β-d-glucans) obtained by the electrostatic sampler were shown significantly higher than those by the BioSampler in most cases. Paired t-tests (n=9) indicated that the sampling difference was statistically significant (p-value <0.05). For allergens, the concentration ratio was up to 5.6, and for toxins the concentration ratio was up to 10.8 when the electrostatic sampler was operated at 1.25 kV/cm and 5 L/min. In general, the concentration ratio decreased for both allergens and toxins when the electrostatic field strength decreased or when the sampling flow rate increased for the electrostatic sampler. Paired t-tests (n=9) also indicated there were statistically significant effects of electrostatic field strength and the sampling flow rates on the performance of the electrostatic sampler when collecting the airborne allergens and toxins.This study presented an airborne environmental allergen and toxin monitoring technology, which holds broad promise for detecting and quantifying low level environmental allergen and toxins.     

Ring-opening copolymerization of α-chloromethyl-α-methyl-β-propiolactone with 1,3-trimethylene carbonate

Summary α-Chloromethyl-α-methyl-β-propiolactone (CMMPL) has been copolymerized with 1,3-trimethylene carbonate (TMC) using a wide range of feed composition and 1,3-dichlorotetrabutyl-distannoxane as a catalyst. Random copolymer, P(CMMPL-co-TMC), was obtained and characterized by ¹H NMR and DSC. The pendant chloromethyl groups of the copolymers are expected to be further modified by reaction with a tertiary amine containing compounds to increase the hydrophilicity of the copolymer or to conjugate bio-active residues onto the copolymer. Received: 24 April 1999/Revised version: 7 May 1999/Accepted: 11 May 1999     

Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein–Protein Interactions of BRCA1 tBRCT Domain

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure–activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein–protein interactions.     

Adsorption to the Surface of Hemozoin Crystals: Structure-Based Design and Synthesis of Amino-Phenoxazine β-Hematin Inhibitors

In silico adsorption of eight antimalarials that inhibit β-hematin (synthetic hemozoin) formation identified a primary binding site on the (001) face, which accommodates inhibitors via formation of predominantly π-π interactions. A good correlation (r²=0.64, P=0.017) between adsorption energies and the logarithm of β-hematin inhibitory activity was found for this face. Of 53 monocyclic, bicyclic and tricyclic scaffolds, the latter yielded the most favorable adsorption energies. Five new amino-phenoxazine compounds were pursued as β-hematin inhibitors based on adsorption behaviour. The 2-substituted phenoxazines show good to moderate β-hematin inhibitory activity (<100 μM) and Plasmodium falciparum blood stage activity against the 3D7 strain. N¹,N¹-diethyl-N⁴-(10H-phenoxazin-2-yl)pentane-1,4-diamine ( P2a ) is the most promising hit with IC₅₀ values of 4.7±0.6 and 0.64±0.05 μM, respectively. Adsorption energies are predictive of β-hematin inhibitory activity, and thus the in silico approach is a beneficial tool for structure-based development of new non-quinoline inhibitors.     

Discovery of Potent Inhibitors of Cyclin-Dependent Kinases 7 and 9: Design,Synthesis, Structure-Activity Relationship Analysis and Biological Evaluation

Cyclin-dependent kinases (CDKs) 7 and 9 are deregulated in various types of human cancer and are thus viewed as therapeutic targets. Accordingly, small-molecule inhibitors of both CDKs are highly sought-after. Capitalising on our previous discovery of CDKI-73, a potent CDK9 inhibitor, medicinal chemistry optimisation was pursued. A number of N-pyridinylpyrimidin-2-amines were rationally designed, chemically synthesised and biologically assessed. Among them, N-(6-(4-cyclopentylpiperazin-1-yl)pyridin-3-yl)-4-(imidazo[1,2-a]pyrimidin-3-yl)pyrimidin-2-amine was found to be one of the most potent inhibitors of CDKs 7 and 9 as well as the most effective anti-proliferative agent towards multiple human cancer cell lines. The cellular mode of action of this compound was investigated in MV4-11 acute myeloid leukaemia cells, revealing that the compound dampened the kinase activity of cellular CDKs 7 and 9, arrested the cell cycle at sub-G1 phase and induced apoptosis.