Metallo‐β‐lactamases (MBLs) cause resistance of Gram‐negative bacteria to β‐lactam antibiotics and are of serious concern, because they can inactivate the last‐resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4‐amino‐2,4‐dihydro‐5‐(2‐methylphenyl)‐3H‐1,2,4‐triazole‐3‐thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non‐amino compound IIIB (1,2‐dihydro‐5‐(2‐methylphenyl)‐3H‐1,2,4‐triazole‐3‐thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3 D structures suggested that the triazole–thione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC₅₀ values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM‐4, VIM‐2, NDM‐1, and IMP‐1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM‐1, VIM‐2, and IMP‐1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc‐dependent hydrolases belonging to the MBL‐fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC₅₀ values toward plasmodial glyoxalase II were in the 10 μm range.

The β‐secretase BACE1 is one of the enzymes that contribute to the production of the Aβ peptide, in vitro and in vivo. JMV1195 was previously shown to inhibit BACE activity in vitro but was unable to block cellular BACE activity. We have designed a new permeable inhibitor, JMV2764 that corresponds to a derivative of JMV1195 to which a penetratin sequence had been added at its N‐terminus. We have assessed the ability of JMV2764 to affect BACE1 activity in vitro, and to modify Aβ production in various cell systems. Endogenous β‐secretase or BACE1 activities were monitored in vitro by means of two distinct fluorimetric substrates in HEK293 extracts of cells expressing either wild‐type βAPP, Swedish mutated βAPP or SPA4CT constructs. Aβ40 recovery was monitored by immunoprecipitation and Western blot analysis. JMV2764 and JMV1195 inhibited endogenous β‐secretase activity of HEK293 cellular homogenates with IC₅₀s of 0.8 and 6.6 μM, respectively. Interestingly, JMV2764 also inhibited β‐secretase activity after preincubation with intact cells while JMV1195 was inactive, indicating that unlike JMV1195, JMV2764 could penetrate into the cells. JMV2764 but not JMV1195 also prevented Aβ production by HEK293 cells overexpressing wild‐type and Swedish‐mutated βAPP. However, JMV2764 was unable to affect Aβ production from cells expressing SPA4CT, a βAPP‐derived sequence that does not need β‐secretase to produce Aβ. Altogether, we have designed a new cell‐permeable BACE1 inhibitor that allows to envision to prevent Aβ production in vivo. Work is in progress to assess the potential of these compounds to prevent Aβ production in transgenic mice overproducing Aβ. British Journal of Pharmacology (2005) 145, 228–235. doi:10.1038/sj.bjp.0706183

To fight the increasingly worrying bacterial resistance to antibiotics, the discovery and development of new therapeutics is urgently needed. Here, we report on a new series of 1,2,4-triazole-3-thione compounds as inhibitors of metallo-β-lactamases (MBLs), which represent major resistance determinants to β-lactams, and especially carbapenems, in Gram-negative bacteria. These molecules are stable analogs of 4-amino-1,2,4-triazole-derived Schiff bases, where the hydrazone-like bond has been reduced (hydrazine series) or the 4-amino group has been acylated (hydrazide series); the synthesis and physicochemical properties thereof are described. The inhibitory potency was determined on the most clinically relevant acquired MBLs (IMP-, VIM-, and NDM-types subclass B1 MBLs). When compared with the previously reported hydrazone series, hydrazine but not hydrazide analogs showed similarly potent inhibitory activity on VIM-type enzymes, especially VIM-2 and VIM-4, with K_i values in the micromolar to submicromolar range. One of these showed broad-spectrum inhibition as it also significantly inhibited VIM-1 and NDM-1. Restoration of β-lactam activity in microbiological assays was observed for one selected compound. Finally, the binding to the VIM-2 active site was evaluated by isothermal titration calorimetry and a modeling study explored the effect of the linker structure on the mode of binding with this MBL.

Amyloid‐β (Aβ) peptides production is thought to be a key event in the neurodegenerative process ultimately leading to Alzheimer’s disease (AD) pathology. A bulk of studies concur to propose that the C‐terminal moiety of Aβ is released from its precursor β‐amyloid precursor protein by a high molecular weight enzymatic complex referred to as γ‐secretase, that is composed of at least, nicastrin (NCT), Aph‐1, Pen‐2, and presenilins (PS) 1 or 2. They are thought to harbor the γ‐secretase catalytic activity. However, several lines of evidence suggest that additional γ‐secretase‐like activities could potentially contribute to Aβ production. By means of a quenched fluorimetric substrate (JMV2660) mimicking the β‐amyloid precursor protein sequence targeted by γ‐secretase, we first show that as expected, this probe allows monitoring of an activity detectable in several cell systems including the neuronal cell line telencephalon specific murine neurons (TSM1). This activity is reduced by DFK167, N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), and LY68458, three inhibitors known to functionally interact with PS. Interestingly, JMV2660 but not the unrelated peptide JMV2692, inhibits Aβ production in an in vitroγ‐secretase assay as expected from a putative substrate competitor. This activity is enhanced by PS1 and PS2 mutations known to be responsible for familial forms of AD and reduced by aspartyl mutations inactivating PS or in cells devoid of PS or NCT. However, we clearly establish that residual JMV2660‐hydrolysing activity could be recovered in PS‐ and NCT‐deficient fibroblasts and that this activity remained inhibited by DFK167. Overall, our study describes the presence of a proteolytic activity displaying γ‐secretase‐like properties but independent of PS and still blocked by DFK167, suggesting that the PS‐dependent complex could not be the unique γ‐secretase activity responsible for Aβ production and delineates PS‐independent γ‐secretase activity as a potential additional therapeutic target to fight AD pathology.