Controlled Production of Amyloid β Peptide from a Photo‐Triggered,Water‐Soluble Precursor “Click Peptide“

In biological experiments, poor solubility and uncontrolled assembly of amyloid β peptide (Aβ) 1–42 pose significant obstacles to establish an experiment system that clarifies the function of Aβ1–42 in Alzheimer's disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water‐soluble photo‐“click peptide” with a coumarin‐derived photocleavable protective group that is based on an O‐acyl isopeptide method. The click peptide had nearly 100‐fold higher water solubility than Aβ1–42 and did not self‐assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Aβ1–42. Moreover, the click peptide afforded Aβ1–42 quickly under physiological conditions (pH 7.4, 37 °C) by photoirradiation followed by an O–N intramolecular acyl migration. Because the in situ production of intact Aβ1–42 from the click peptide could improve the difficulties in handling Aβ1–42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Aβ1–42 in AD.     

Retro‐Enantio N‐Methylated Peptides as β‐Amyloid Aggregation Inhibitors

An emerging and attractive target for the treatment of Alzheimer's disease is to inhibit the aggregation of β‐amyloid protein (Aβ). We applied the retro‐enantio concept to design an N‐methylated peptidic inhibitor of the Aβ42 aggregation process. This inhibitor, inrD, as well as the corresponding all‐L (inL) and all‐D (inD) analogues were assayed for inhibition of Aβ42 aggregation. They were also screened in neuroblastoma cell cultures to assess their capacity to inhibit Aβ42 cytotoxicity and evaluated for proteolytic stability. The results reveal that inrD and inD inhibit Aβ42 aggregation more effectively than inL, that inrD decreases Aβ42 cytotoxicity to a greater extent than inL and inD, and that as expected, both inD and inrD are stable to proteases. Based on these results, we propose that the retro‐enantio approach should be considered in future designs of peptide inhibitors of protein aggregation.     

Copper and Zinc Binding to Amyloid‐β: Coordination,Dynamics, Aggregation,Reactivity and Metal‐Ion Transfer

The metal ions copper, zinc and iron have been shown to be involved in Alzheimer's disease (AD). Cu, Zn and Fe ions are proposed to be implicated in two key steps of AD pathology: 1) aggregation of the peptide amyloid‐β (Aβ), and 2) production of reactive oxygen species (ROS) induced by Aβ. There is compelling evidence that Cu and Zn bind directly to Aβ in AD. This formation of Cu/Zn–Aβ complexes is thought to be aberrant as they have been detected only in AD, but not under healthy conditions. In this context, the understanding of how these metal ions interact with Aβ, their influence on structure and oligomerization become an important issue for AD. Moreover, the mechanism of ROS production by Cu–Aβ in relation to its aggregations state, as well as the metal‐transfer reaction from and to Aβ are crucial in order to understand why Aβ oligomers are highly toxic and why Aβ seems to bind Cu and Zn only in AD.     

Identification of Physiological and Toxic Conformations in Aβ42 Aggregates

Aggregation of the 42‐residue amyloid β‐protein (Aβ42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Despite numerous structural studies on Aβ aggregates, the relationship between tertiary structure and toxicity remains unclear. Our proline scanning and solid‐state NMR studies suggested that aggregates both of wild‐type Aβ42 and of E22K‐Aβ42 (one of the mutants related to cerebral amyloid angiopathy) contain two conformers: a major one with a turn at positions 25 and 26, and a minor one with a turn at positions 22 and 23. To identify the toxic conformer, the derivative Aβ42‐lactam(22K–23E), in which the side chains at positions 22 and 23 were covalently linked, was synthesized as a minor conformer surrogate, along with Aβ42‐lactam(25K–26E) as a major conformer surrogate. The Aβ42‐lactam(22K–23E) showed stronger aggregation, neurotoxicity, radical generation, and oligomerization than wild‐type Aβ42, whereas in Aβ42‐lactam(25K–26E) were weak. The transition from the physiological conformation with a turn at positions 25 and 26 to the toxic conformation with a turn at positions 22 and 23 might be a key event in the pathogenesis of AD.     

Controlling Amyloid‐β Peptide(1–42) Oligomerization and Toxicity by Fluorinated Nanoparticles

The amyloid‐β peptide (Aβ) is a major fibrillar component of neuritic plaques in Alzheimer's disease brains and is related to the pathogenesis of the disease. Soluble oligomers that precede fibril formation have been proposed as the main neurotoxic species that contributes to neurodegeneration and dementia. We hypothesize that oligomerization and cytotoxicity can be repressed by nanoparticles (NPs) that induce conformational changes in Aβ42. We show here that fluorinated and hydrogenated NPs with different abilities to change Aβ42 conformation influence oligomerization as assessed by atomic force microscopy, immunoblot and SDS‐PAGE. Fluorinated NPs, which promote an increase in α‐helical content, exert an antioligomeric effect, whereas hydrogenated analogues do not and lead to aggregation. Cytotoxicity assays confirmed our hypothesis by indicating that the conformational conversion of Aβ42 into an α‐helical‐enriched secondary structure also has antiapoptotic activity, thereby increasing the viability of cells treated with oligomeric species.     

Copper inducing Aβ42 rather than Aβ40 nanoscale oligomer formation is the key process for Aβ neurotoxicity

Copper is known to be a critical factor in Alzheimer's disease (AD) pathogenesis, as it is involved in amyloid-β (Aβ) peptide related toxicity. However, the relationship between neurotoxicity and Aβ peptide in the presence of copper remains unclear. The effect of copper has not been clearly differentiated between Aβ42 and Aβ40, and it is still debated whether copper-mediated neurotoxicity is due to reactive oxygen species (ROS) accumulation or other molecular mechanisms. Here, we describe that copper dramatically affects Aβ42 aggregation and enhances Aβ42 cytotoxicity while it shows no significant effects on Aβ40. These phenomena are mainly because that the strong interactions between copper and Aβ42 lead to great conformation changes, and stabilize Aβ42 aggregates at highly toxic nanoscale oligomer stage, whereas copper shows no similar impact on Aβ40. We also propose a possible molecular mechanism that copper enhances Aβ42 cytotoxicity via perturbing membrane structure. Moreover, we test the effect of an analogue of copper, nickel, on Aβ aggregation and cytotoxicity, finding that nickel also enhances cytotoxicity via Aβ42 nanoscale oligomer formation. These results clarify that the copper-induced Aβ42 nanoscale oligomer formation is the key process for Aβ neurotoxicity, and suggest that disrupting the interactions between copper and Aβ42 peptide to inhibit nanoscale oligomerization process, deserves more attention in AD drug development.     

Copper(II)‐bis‐Histidine Coordination Structure in a Fibrillar Amyloid β‐Peptide Fragment and Model Complexes Revealed by Electron Spin Echo Envelope Modulation Spectroscopy

Truncated and mutated amyloid‐β (Aβ) peptides are models for systematic study—in homogeneous preparations—of the molecular origins of metal ion effects on Aβ aggregation rates, types of aggregate structures formed, and cytotoxicity. The 3D geometry of bis‐histidine imidazole coordination of Cu^II in fibrils of the nonapetide acetyl‐Aβ(13–21)H14A has been determined by powder ¹⁴N electron spin echo envelope modulation (ESEEM) spectroscopy. The method of simulation of the anisotropic combination modulation is described and benchmarked for a Cu^II‐bis‐cis‐imidazole complex of known structure. The revealed bis‐cis coordination mode, and the mutual orientation of the imidazole rings, for Cu^II in Ac‐Aβ(13–21)H14A fibrils are consistent with the proposed β‐sheet structural model and pairwise peptide interaction with Cu^II, with an alternating [‐metal‐vacancy‐]_n pattern, along the N‐terminal edge. Metal coordination does not significantly distort the intra‐β‐strand peptide interactions, which provides a possible explanation for the acceleration of Ac‐Aβ(13–21)H14A fibrillization by Cu^II, through stabilization of the associated state and low‐reorganization integration of β‐strand peptide pair precursors.     

Scouting around 1,2,3,4-Tetrahydrochromeno[3,2-c]pyridin-10-ones for Single- and Multitarget Ligands Directed towards Relevant Alzheimer's Targets

A number of 1,2,3,4-tetrahydrochromeno[3,2-c]pyridin-10-one derivatives have been synthesized and screened against different targets involved in the onset and progression of Alzheimer's disease (AD), such as acetyl- and butyrylcholinesterase (AChE and BChE), monoamine oxidases A and B (MAO A and B), aggregation of β-amyloid (Aβ) and reactive oxygen species (ROS) production. Derivatives 1 c , 3 b , 4 and 5 a showed multifaceted profiles of promising anti-AD features and returned well-balanced multitargeting inhibitory activities. Moreover, compound 1 f , a potent and selective human MAO B inhibitor (IC₅₀=0.89 μM), proved to be a safe neuroprotectant in a human neuroblastoma cell line (SH-SY5Y) by improving viability impaired by Aβ_1–42 and pro-oxidant insult. Furthermore, structure–activity relationships (SARs) and docking models were derived in order to assist further hit-to-lead optimization stage.     

Synthesis of New Tyrosol-Based Phosphodiester Derivatives: Effect on Amyloid β Aggregation and Metal Chelation Ability

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. Increasing evidence has shown that aggregation of amyloid β (Aβ) and oxidative stress are strictly interconnected, and their modulation might have a positive and synergic effect in contrasting AD-related impairments. Herein, a new and efficient fragment-based approach towards tyrosol phosphodiester derivatives (TPDs) has been developed starting from suitable tyrosol building blocks and exploiting the well-established phosphoramidite chemistry. The antioxidant activity of new TPDs has been tested as well as their ability to inhibit Aβ protein aggregation. In addition, their metal chelating ability has been evaluated as a possible strategy to develop new natural-based entities for the prevention or therapy of AD. Interestingly, TPDs containing a catechol moiety have demonstrated highly promising activity in inhibiting the aggregation of Aβ₄₀ and a strong ability to chelate biometals such as Cu^II and Zn^II.     

Copper Transfer from Cu–Aβ to Human Serum Albumin Inhibits Aggregation,Radical Production and Reduces Aβ Toxicity

Amyloid‐β peptides (Aβ) and the protein human serum albumin (HSA) interact in vivo. They are both localised in the blood plasma and in the cerebrospinal fluid. Among other functions, HSA is involved in the transport of the essential metal copper. Complexes between Aβ and copper ions have been proposed to be an aberrant interaction implicated in the development of Alzheimer's disease, where Cu is involved in Aβ aggregation and production of reactive oxygen species (ROS). In the present work, we studied copper‐exchange reaction between Aβ and HSA or the tetrapeptide DAHK (N‐terminal Cu‐binding domain of HSA) and the consequence of this exchange on Aβ‐induced ROS production and cell toxicity. The following results were obtained: 1) HSA and DAHK removed Cu^II from Aβ rapidly and stoichiometrically, 2) HSA and DAHK were able to decrease Cu‐induced aggregation of Aβ, 3) HSA and DAHK suppressed the catalytic HO^. production in vitro and ROS production in neuroblastoma cells generated by Cu–Aβ and ascorbate, 4) HSA and DAHK were able to rescue these cells from the toxicity of Cu–Aβ with ascorbate, 5) DAHK was more potent in ROS suppression and restoration of neuroblastoma cell viability than HSA, in correlation with an easier reduction of Cu^II–HSA than Cu–DAHK by ascorbate, in vitro. Our data suggest that HSA is able to decrease aberrant Cu^II–Aβ interaction. The repercussion of the competition between HSA and Aβ to bind Cu in the blood and brain and its relation to Alzheimer's disease are discussed.     

Novel Tacrine‐Grafted Ugi Adducts as Multipotent Anti‐Alzheimer Drugs: A Synthetic Renewal in Tacrine–Ferulic Acid Hybrids

Herein we describe the design, multicomponent synthesis, and biological, molecular modeling and ADMET studies, as well as in vitro PAMPA‐blood–brain barrier (BBB) analysis of new tacrine–ferulic acid hybrids (TFAHs). We identified (E)‐3‐(hydroxy‐3‐methoxyphenyl)‐N‐{8[(7‐methoxy‐1,2,3,4‐tetrahydroacridin‐9‐yl)amino]octyl}‐N‐[2‐(naphthalen‐2‐ylamino)2‐oxoethyl]acrylamide (TFAH 10 n ) as a particularly interesting multipotent compound that shows moderate and completely selective inhibition of human butyrylcholinesterase (IC₅₀=68.2 nM ), strong antioxidant activity (4.29 equiv trolox in an oxygen radical absorbance capacity (ORAC) assay), and good β‐amyloid (Aβ) anti‐aggregation properties (65.6 % at 1:1 ratio); moreover, it is able to permeate central nervous system (CNS) tissues, as determined by PAMPA‐BBB assay. Notably, even when tested at very high concentrations, TFAH 10 n easily surpasses the other TFAHs in hepatotoxicity profiling (59.4 % cell viability at 1000 μM ), affording good neuroprotection against toxic insults such as Aβ_1–40, Aβ_1–42, H₂O₂, and oligomycin A/rotenone on SH‐SY5Y cells, at 1 μM . The results reported herein support the development of new multipotent TFAH derivatives as potential drugs for the treatment of Alzheimer′s disease.     

Quantifying Prefibrillar Amyloids in vitro by Using a “Thioflavin‐Like” Spectroscopic Method

In Alzheimer's disease (AD) and other neurodegenerative disorders, proteins accumulate into ordered aggregates, called amyloids. Recent evidence suggests that these structures include both large, insoluble fibrils and smaller, prefibrillar structures, such as dimers, oligomers, and protofibrils. Recently, focus has shifted to the prefibrillar aggregates because they are highly neurotoxic and their levels appear to correlate with cognitive impairment. Thus, there is interest in finding methods for specifically quantifying these structures. One of the classic ways of detecting amyloid formation is through the fluorescence of the benzothiazole dye, thioflavin T (ThT). This reagent has been a “workhorse” of the amyloid field because it is robust and inexpensive. However, one of its limitations is that it does not distinguish between prefibrillar and fibrillar aggregates. We screened a library of 37 indoles for those that selectively change fluorescence in the presence of prefibrillar amyloid‐β (Aβ). From this process, we selected the most promising example, tryptophanol (TROL), to use in a quantitative “thioflavin‐like” assay. Using this probe in combination with electron microscopy, we found that prefibrils are largely depleted during Aβ aggregation in vitro but that they remain present after the apparent saturation of the ThT signal. These results suggest that a combination of TROL and ThT provides greater insight into the process of amyloid formation by Aβ. In addition, we found that TROL also recognizes other amyloid‐prone proteins, including ataxin‐3, amylin, and CsgA. Thus, this assay might be an inexpensive spectroscopic method for quantifying amyloid prefibrils in vitro.     

Benzofuran–Chalcone Hybrids as Potential Multifunctional Agents against Alzheimer’s Disease: Synthesis and in vivo Studies with Transgenic Caenorhabditis elegans

In the search for effective multifunctional agents for the treatment of Alzheimer’s disease (AD), a series of novel hybrids incorporating benzofuran and chalcone fragments were designed and synthesized. These hybrids were screened by using a transgenic Caenorhabditis elegans model that expresses the human β‐amyloid (Aβ) peptide. Among the hybrids investigated, (E)‐3‐(7‐methyl‐2‐(4‐methylbenzoyl)benzofuran‐5‐yl)‐1‐phenylprop‐2‐en‐1‐one ( 4 f ), (E)‐3‐(2‐benzoyl‐7‐methylbenzofuran‐5‐yl)‐1‐phenylprop‐2‐en‐1‐one ( 4 i ), and (E)‐3‐(2‐benzoyl‐7‐methylbenzofuran‐5‐yl)‐1‐(thiophen‐2‐yl)prop‐2‐en‐1‐one ( 4 m ) significantly decreased Aβ aggregation and increased acetylcholine (ACh) levels along with the overall availability of ACh at the synaptic junction. These compounds were also found to decrease acetylcholinesterase (AChE) levels, reduce oxidative stress in the worms, lower lipid content, and to provide protection against chemically induced cholinergic neurodegeneration. Overall, the multifunctional effects of these hybrids qualify them as potential drug leads for further development in AD therapy.     

Single‐Molecule Imaging Reveals that Small Amyloid‐β1–42 Oligomers Interact with the Cellular Prion Protein (PrPC)

Oligomers of the amyloid‐β peptide (Aβ) play a central role in the pathogenesis of Alzheimer’s disease and have been suggested to induce neurotoxicity by binding to a plethora of cell‐surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single‐molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrP^C) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrP^C and that the species bound to PrP^C are predominantly small oligomers (dimers and trimers). Single‐molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor‐mediated oAβ‐induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.     

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid β Aggregation

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid β (Aβ) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aβ deposits that not only modulates Aβ aggregation, but also acts as a receptor for Aβ fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aβ could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aβ fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.     

Real-Time BODIPY-Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1–42 Peptide

Misfolding and aggregation of amyloid β1–42 peptide (Aβ1–42) play a central role in the pathogenesis of Alzheimer's disease (AD). Targeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents a promising therapeutic strategy to reduce the toxicity associated with Aβ1–42. Currently, the thioflavin T (ThT) assay is the only established spectrofluorometric method to screen aggregation inhibitors. The success of the ThT assay is that it can detect Aβ1–42 aggregates with high β-sheet content, such as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately, by using the ThT assay, the detection of inhibitors of early soluble oligomers that present a low β-sheet character is challenging. Herein, a new, facile, and robust boron-dipyrromethene (BODIPY) real-time assay suitable for 96-well plate format, which allows screening of compounds as selective inhibitors of the formation of Aβ1–42 oligomers, is reported. These inhibitors decrease the cellular toxicity of Aβ1–42, although they fail in the ThT assay. The findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is a convenient method to screen and discover new candidate compounds that slow down or stop the pathological early oligomerization process and are active in the cellular assay. Therefore, it is a suitable complementary screening method of the current ThT assay.     

Micellar and Interfacial Behavior of Mixed Systems Containing Glycoside‐Based Surfactant and Cationic Didecyldimethylammonium Chloride

The behavior of binary mixtures of glycoside‐based surfactants in combination with didecyldimethylammonium chloride (DDAC) has been studied using surface tension measurements. The three glycoside‐based surfactants are nonionic decyl glycoside (APG), nonionic dodecyl ethoxy glycoside (AEG) and anionic disodium dodecyl ethoxy glycoside citrate (AEG‐EC). Lower critical micelle concentrations (CMC) and minimum molecular area (A_min) values were obtained for all the mixed systems. The pC₂₀ values of APG/DDAC and AEG‐EC/DDAC mixtures are larger than pure surfactants, while the values of AEG/DDAC are between those of AEG and DDAC. Interactions between the monomers have also been investigated by determining the interaction parameters. Negative β_m and β_s values indicate synergistic effects in both the mixed micelle and monolayer formation. For different mixed systems, interaction in the mixed micelle formation becomes stronger in the order: AEG/DDAC < AEG‐EC/DDAC < APG/DDAC. The degree of synergism in the mixed monolayer formation follows the order: AEG/DDAC < APG/DDAC < AEG‐EC/DDAC.     

Multitarget Therapeutic Leads for Alzheimer’s Disease: Quinolizidinyl Derivatives of Bi‐ and Tricyclic Systems as Dual Inhibitors of Cholinesterases and β‐Amyloid (Aβ) Aggregation

Multitarget therapeutic leads for Alzheimer’s disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting β‐amyloid (Aβ) oligomerization. Thirty‐seven thioxanthen‐9‐one, xanthen‐9‐one, naphto‐ and anthraquinone derivatives were tested for the direct inhibition of Aβ(1–40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi‐ and tri‐cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of β‐amyloid. In most cases, IC₅₀ values were in the low micromolar and sub‐micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC₅₀=0.84 μM ) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and Aβ aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics.     

Carnosine Inhibits Aβ42 Aggregation by Perturbing the H‐Bond Network in and around the Central Hydrophobic Cluster

Aggregation of the amyloid‐β peptide (Aβ) into fibrillar structures is a hallmark of Alzheimer's disease. Thus, preventing self‐assembly of the Aβ peptide is an attractive therapeutic strategy. Here, we used experimental techniques and atomistic simulations to investigate the influence of carnosine, a dipeptide naturally occurring in the brain, on Aβ aggregation. Scanning force microscopy, circular dichroism and thioflavin T fluorescence experiments showed that carnosine does not modify the conformational features of Aβ42 but nonetheless inhibits amyloid growth. Molecular dynamics (MD) simulations indicated that carnosine interacts transiently with monomeric Aβ42 by salt bridges with charged side chains, and van der Waals contacts with residues in and around the central hydrophobic cluster (¹⁷LVFFA²¹). NMR experiments on the nonaggregative fragment Aβ12–28 did not evidence specific intermolecular interactions between the peptide and carnosine, in agreement with MD simulations. However, a close inspection of the spectra revealed that carnosine interferes with the local propensity of the peptide to form backbone hydrogen bonds close to the central hydrophobic cluster (residues E22, S26 and N27). Finally, MD simulations of aggregation‐prone Aβ heptapeptide segments show that carnosine reduces the propensity to form intermolecular backbone hydrogen bonds in the region 18–24. Taken together, the experimental and simulation results (cumulative MD sampling of 0.2 ms) suggest that, despite the inability of carnosine to form stable contacts with Aβ, it might block the pathway toward toxic aggregates by perturbing the hydrogen bond network near residues with key roles in fibrillogenesis.     

Diaryl‐Substituted Azolylthioacetamides: Inhibitor Discovery of New Delhi Metallo‐β‐Lactamase‐1 (NDM‐1)

The emergence and spread of antibiotic‐resistant pathogens is a global public health problem. Metallo‐β‐lactamases (MβLs) such as New Delhi MβL‐1 (NDM‐1) are principle contributors to the emergence of resistance because of their ability to hydrolyze almost all known β‐lactam antibiotics including penicillins, cephalosporins, and carbapenems. A clinical inhibitor of MBLs has not yet been found. In this study we developed eighteen new diaryl‐substituted azolylthioacetamides and found all of them to be inhibitors of the MβL L1 from Stenotrophomonas maltophilia (K_i<2 μM ), thirteen to be mixed inhibitors of NDM‐1 (K_i<7 μM ), and four to be broad‐spectrum inhibitors of all four tested MβLs CcrA from Bacteroides fragilis, NDM‐1 and ImiS from Aeromonas veronii, and L1 (K_i<52 μM ), which are representative of the B1a, B1b, B2, and B3 subclasses, respectively. Docking studies revealed that the azolylthioacetamides, which have the broadest inhibitory activity, coordinate to the Zn^II ion(s) preferentially via the triazole moiety, while other moieties interact mostly with the conserved active site residues Lys224 (CcrA, NDM‐1, and ImiS) or Ser221 (L1).