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The increasing prevalence of metallo‐β‐lactamase (MBL)‐expressing bacteria presents a worrying trend in antibiotic resistance. MBLs rely on active site zinc ions for their hydrolytic activity and the pursuit of MBL‐inhibitors has therefore involved the investigation of zinc chelators. To ensure that such chelators specifically target MBLs, a series of cephalosporin prodrugs of two potent zinc‐binders: dipicolinic acid (DPA) and 8‐thioquinoline (8‐TQ) was prepared. Although both DPA and 8‐TQ bind free zinc very tightly (K d values in the low nm range), the corresponding cephalosporin conjugates do not. The cephalosporin conjugates are efficiently hydrolyzed by MBLs to release DPA or 8‐TQ, as confirmed by using both NMR and LC‐MS studies. Notably, the cephalosporin prodrugs of DPA and 8‐TQ show potent inhibitory activity against NDM, VIM, and IMP classes of MBLs and display potent synergy with meropenem against MBL‐expressing clinical isolates of K. pneumoniae and E. coli., Stopping bacterial resistance: Metallo‐β‐lactamases (MBLs) are bacterial resistance enzymes that degrade β‐lactam antibiotics, including last line of defense carbapenems like meropenem. A new series of cephalosporin prodrug inhibitors that exploit the hydrolytic action of the target MBL itself to activate the inhibitor is reported. The prodrugs display potent MBL inhibition and effectively synergize with meropenem against highly resistant MBL‐expressing isolates.

Nanobodies have recently been introduced to the field of photodynamic therapy (PDT) as a very promising strategy to target photosensitizers selectively to cancer cells. Nanobodies are known for their characteristic small size (15 kDa), high specificity, and high binding affinities. These features allow rapid accumulation of nanobody-photosensitizer conjugates at the tumor site and rapid clearance of unbound fractions, and thus illumination for activation is possible 1 or 2 h postinjection. Preclinical studies have shown extensive tumor damage after nanobody-targeted PDT . This chapter addresses the first steps toward preparing nanobody-photosensitizer conjugates, which are the nanobody production and purification. The protocol for nanobody production addresses either medium- or large-scale bacterial expression, while the nanobody purification is described for two main strategies: affinity chromatography and ion-exchange chromatography. For the first strategy, protocols are described for different affinity tags and purification from either medium-scale or large-scale productions. For the second strategy, the protocol given is for purification from a large-scale production.

Fluorophores have been conjugated to nanobodies for approximately a decade, for several applications in molecular biology. More recently, photosensitizers have been conjugated to nanobodies for targeted photodynamic therapy (PDT). The most common chemistry is the random conjugation in which commercial fluorophores or photosensitizers contain a N-hydroxysuccinimide ester (NHS ester) group that reacts specifically and efficiently with lysines in the amino acid sequence of the nanobody and with the N-terminal amino groups to form a stable amide bond. Alternatively, maleimide-containing fluorophores or photosensitizers can be used for conjugation to thiols, in a site-directed manner through a cysteine incorporated at the C-terminal of the nanobody. This chapter addresses both conjugation strategies, providing details on the reaction conditions, purification, and characterization of the conjugates obtained.

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., Developing broad‐spectrum MBL inhibitors: Structurally diverse MBL inhibitors were assessed for their ability to inhibit purified MBL enzymes, bind various divalent cations, and resensitize a carbapenem‐resistant E. coli strain to meropenem. These studies reveal that potent MBL inhibition and synergy with meropenem is not explicitly dependent on the ability of an inhibitor to bind free zinc with high affinity.

In an attempt to exploit the hydrolytic mechanism by which β-lactamases degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-β-lactamases (MBLs) in a spatiotemporally controlled fashion. While enzymatic hydrolysis of the β-lactam ring was observed, it was not accompanied by inhibitor release. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs. In addition, conjugate 8 was also found to greatly reduce the minimum inhibitory concentration of meropenem against IMP-producing bacteria. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure–activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with Trp28 within the IMP active site may contribute to its potency and selectivity.

In an attempt to exploit the hydrolytic mechanism by which β-lactamase enzymes degrade cephalosporins, we designed and synthesized a series of novel cephalosporin prodrugs aimed at delivering thiol-based inhibitors of metallo-β-lactamases (MBLs) in spatiotemporally controlled fashion. Notably, while enzyme-mediated hydrolysis of the β-lactam ring was found to occur, it was not accompanied by release of the thiol-based inhibitors. Nonetheless, the cephalosporin prodrugs, especially thiomandelic acid conjugate (8), demonstrated potent inhibition of IMP-type MBLs, with IC50 values in the nanomolar range. In addition, conjugate 8 was also found to greatly reduce the MIC of meropenem against an IMP-28 producing clinical isolate of K. pneumoniae. The results of kinetic experiments indicate that these prodrugs inhibit IMP-type MBLs by acting as slowly turned-over substrates. Structure-activity relationship studies revealed that both phenyl and carboxyl moieties of 8 are crucial for its potency. Furthermore, modeling studies indicate that productive interactions of the thiomandelic acid moiety of 8 with residues Trp28 and Lys161 within the IMP active site may contribute to the observed inhibitory potency and selectivity.

Metallo-β-lactamases (MBLs) are zinc-dependent bacterial resistance enzymes that can inactivate essentially all classes of β-lactam antibiotics. Infections due to multi-drug-resistant pathogens that express MBLs are difficult to treat and carry high mortality rates. At present there are no clinically approved MBL inhibitors underscoring the urgent need for pharmaceutical agents capable of counteracting the action of these enzymes. In order to develop effective MBL inhibitors it is essential to understand their inhibitory mechanisms. We here describe a comprehensive mechanistic study on a panel of structurally distinct MBL inhibitors drawn from the diverse collection of compounds reported to date in both the scientific and patent literature. Specifically, we determined the half-maximal inhibitory concentration value (IC50) for each inhibitor against purified NDM-1 and IMP-1 revealing clear differences in inhibitory potency among the compounds tested. Additional mechanistic insights into metal binding were also obtained by means of isothermal titration calorimetry (ITC) which was used to assess the affinity of the MBL inhibitors for Zn2+, Ca2+ and Mg2+. These investigations revealed clear differences in metal binding among the MBL inhibitors evaluated. In addition, we directly compared the ability of these compounds to resensitize an NDM-1-expressing E. coli strain to the last resort carbapenem antibiotic meropenem. Notably, indole carboxylate 12 proved to be the most potent inhibitor tested in its ability to synergize with meropenem and with IC50 values in the low nanomolar range against both enzymes. Interestingly, while compound 12 was found the most active MBL inhibitor, it exhibited no appreciable binding to any of the metals tested. These findings provide valuable insights into differences in mechanism and potency for the various classes of MBL inhibitors reported to date.

In the search for new inhibitors of bacterial metallo-β-lactamases (MBLs), a series of commonly used small molecule carboxylic acid derivatives were evaluated for their ability to inhibit New Delhi metallo-β-lactamase (NDM)-, Verona integron-encoded metallo-β-lactamase (VIM)-, and imipenemase (IMP)-type enzymes. Nitrilotriacetic acid (3) and N-(phosphonomethyl)iminodiacetic acid (5) showed promising activity especially against NDM-1 and VIM-2 with IC50 values in the low-to-sub μM range. Binding assays using isothermal titration calorimetry reveal that 3 and 5 bind zinc with high affinity with dissociation constant (Kd) values of 121 and 56 nM, respectively. The in vitro biological activity of 3 and 5 against E. coli expressing NDM-1 was evaluated in checkerboard format, demonstrating a strong synergistic relationship for both compounds when combined with Meropenem. Compounds 3 and 5 were then tested against 35 pathogenic strains expressing MBLs of the NDM, VIM, or IMP classes. Notably, when combined with Meropenem, compounds 3 and 5 were found to lower the minimum inhibitory concentration (MIC) of Meropenem up to 128-fold against strains producing NDM- and VIM-type enzymes.

A series of aminocarboxylic acid analogues of aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) were chemoenzymatically synthesized via the addition of various mono- and diamine substrates to fumaric acid catalyzed by the enzyme EDDS lyase. Many of these novel AMA and EDDS analogues demonstrate potent inhibition of the bacterial metallo-β-lactamase NDM-1. Isothermal titration calorimetry assays revealed a strong correlation between the inhibitory potency of the compounds and their ability to bind zinc. Compounds 1a (AMA), 1b (AMB), 5 (EDDS), followed by 1d and 8a, demonstrate the highest synergy with meropenem resensitizing an NDM-1 producing strain of E. coli to this important carbapenem of last resort.

Isoniazid (INH or isonicotinic hydrazide) is used for the treatment and prophylaxis of tuberculosis. Liver and brain are two important target organs in INH toxicity. However, the exact mechanisms behind the INH hepatotoxicity or neurotoxicity have not yet been completely understood. Considering the mitochondria as one of the possible molecular targets for INH toxicity, the aim of this study was to evaluate the mechanisms of INH mitochondrial toxicity on isolated mitochondria. Mitochondria were isolated by differential ultracentrifugation from male Sprague-Dawley rats and incubated with different concentrations of INH (25-2000 μM) for the investigation of mitochondrial parameters. The results indicated that INH could interact with mitochondrial respiratory chain and inhibit its activity. Our results showed an elevation in mitochondrial reactive oxygen species (ROS) formation, lipid peroxidation and mitochondrial membrane potential collapse after exposure of isolated liver mitochondria in INH. However, different results were obtained in brain mitochondria. Noteworthy, significant glutathione oxidation, adenosine triphosphate (ATP) depletion and lipid peroxidation were observed in higher concentration of INH, as compared to liver mitochondria. In conclusion, our results suggest that INH may initiate its toxicity in liver mitochondria through interaction with electron transfer chain, lipid peroxidation, mitochondrial membrane potential decline and cytochrome c expulsion which ultimately lead to cell death signaling.

Thallium(I) is a highly toxic heavy metal; however, up to now, its mechanisms are poorly understood. The authors' previous studies showed that this compound could induce reactive oxygen species (ROS) formation, reduced glutathione (GSH) oxidation, membrane lipid peroxidation, and mitochondrial membrane potential (MMP) collapse in isolated rat hepatocyte. Because the liver is the storage site of thallium, it seems that the liver mitochondria are one of the important targets for hepatotoxicity. In this investigation, the effects of thallium on mitochondria were studied to investigate its mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with different concentrations of thallium (25–200 µM). Thallium(I)-treated mitochondria showed a marked elevation in oxidative stress parameters accompanied by MMP collapse when compared with the control group. These results showed that different concentrations of thallium (25–200 µM) induced a significant (P

Considering the structural features of a group of known potent inhibitors of human platelet aggregation containing hydrazone structural backbone, a series of novel hydrazone derivatives of 2-hydrazinyl-1,3,4-thiadiazole were synthesized using a one-pot process and tested for their inhibitory activity against platelet aggregation induced by arachidonic acid and ADP. Among the derivatives, compounds 3l, 3o and 3p exhibited the highest antiplatelet aggregation activity. The derivatives were also screened for their potential antimycobacterial activity and compounds 3g, 3k, 3p and 3q were among the most active compounds.

Based on the existing reports regarding the antiplatelet aggregation activity of hydrazone derivatives, a series of indole hydrazone derivatives were considered as potential antiplatelet agents and synthesized. The structures of the synthesized compounds were confirmed by spectral data and elemental analysis. The new indole hydrazone derivatives were evaluated for their ability to inhibit platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid (AA). Compounds 1h and 3h exhibited remarkable activity against arachidonic acid induced platelet aggregation with IC(50) values comparable to that of indomethacin and compound 1i efficiently inhibited platelet aggregation induced by both ADP and AA.

Compounds (IVa,b, d) show antiplatelet aggregation activity comparable to that of indomethacin.

Based on the structural elements of bioactive indole-based compounds, a series of novel 1-substituted indole-3-carboxaldehyde thiosemicarbazones were synthesized as potential antimycobacterial and anticancer agents. The derivatives were prepared via a two-step methodology including N-alkylation(benzylation) of indole-3-carboxaldehyde and conversion of the intermediate aldehydes to corresponding thiosemicarbazones. The derivatives were evaluated for their antimycobacterial activity and compounds 3d (R = propyl) and 3q (R = 4-nitrobenzyl) were among the most potent and selective derivatives with IC50 values of 0.9 and 1.9 μg/mL respectively. The anticancer activity of the derivatives was also assessed against a panel of tumor cell lines. Compounds 3t, 3u, 3v and 3w efficiently inhibited the majority of the cancer cell lines with considerable selectivity.

During the past 10 years, the use of methamphetamine (METH) has significantly increased in Iran and around the world. The widespread use of 3,4-methylenedioxymethamphetamine as a recreational drug has been responsible for the incidence of several cases of liver failure in young people. This issue made researchers focus on METH toxicity due to the lack of effective treatment and human health risk assessment. There are several reports showing that its long-term use increases the risk for dopamine depletion, but the toxicity mechanisms of METH in liver are not well understood. Therefore, we aimed to investigate the mitochondrial toxicity mechanisms of METH on isolated mitochondria. Rat liver mitochondria were obtained by differential ultracentrifugation, and the isolated mitochondria were then incubated with different concentrations of METH (2.5–20 μM). Our results showed that this agent could induce oxidative stress via rising in mitochondrial reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential collapse, and mitochondrial swelling. In addition, collapse of mitochondrial membrane potential, mitochondrial swelling, and release of cytochrome c following METH treatment were well inhibited by pretreatment of mitochondria with cyclosporin A and butylated hydroxytoluene. Finally, it is suggested that METH could interact with respiratory complexes (II and III) and METH-induced liver toxicity may be the result of its disruptive effect on mitochondrial respiratory chain that is the obvious cause of ROS formation, mitochondrial membrane potential decline, and cytochrome c expulsion which start cell death signaling.

Thallium(I) is a highly toxic heavy metal; however, up to now, its mechanisms are poorly understood. The authors' previous studies showed that this compound could induce reactive oxygen species (ROS) formation, reduced glutathione (GSH) oxidation, membrane lipid peroxidation, and mitochondrial membrane potential (MMP) collapse in isolated rat hepatocyte. Because the liver is the storage site of thallium, it seems that the liver mitochondria are one of the important targets for hepatotoxicity. In this investigation, the effects of thallium on mitochondria were studied to investigate its mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with different concentrations of thallium (25-200 µM). Thallium(I)-treated mitochondria showed a marked elevation in oxidative stress parameters accompanied by MMP collapse when compared with the control group. These results showed that different concentrations of thallium (25-200 µM) induced a significant (P 0.05) increase in mitochondrial ROS formation, ATP depletion, GSH oxidation, mitochondrial outer membrane rupture, mitochondrial swelling, MMP collapse, and cytochrome c release. In general, these data strongly supported that the thallium(I)-induced liver toxicity is a result of the disruptive effect of this metal on the mitochondrial respiratory complexes (I, II, and IV), which are the obvious causes of metal-induced ROS formation and ATP depletion. The latter two events, in turn, trigger cell death signaling via opening of mitochondrial permeability transition pore and cytochrome c expulsion.

Background: Perfluorooctanoic acid (PFOA) is one of the most widely used perfluoroalkanes as surfactants, lubricants and processing aids in the production of polymers, which has also been detected in the environment, wildlife and human body. Animal studies indicated that PFOA caused a wide array of toxic effects including liver and brain dysfunction, carcinogenicity and reproductive and developmental toxicity. Based on the established role of mitochondria-mediated pathways in the observed toxic effects of many drugs and chemicals, in this study, the potential toxic effects of PFOA on mitochondria isolated from rat liver and brain have been investigated. Method: Mitochondria were isolated by differential centrifugation method and incubated with different concentrations of PFOA (0.5–1.5 mM). The effects of PFOA were assessed on a series of mitochondrial parameters including reactive oxygen species (ROS) formation, activities of mitochondrial complexes I/II/III, reduced glutathione (GSH) content, adenosine triphosphate (ATP) level, membrane potential, lipid peroxidation (LPO), mitochondrial swelling and cytochrome c release. Results: The data on liver mitochondria indicated that PFOA-induced ROS elevation in both mitochondrial complexes I and III, mitochondrial membrane potential collapse, swelling, cytochrome c release and decreased ATP level which induces apoptosis or necrosis. On brain mitochondria, PFOA showed fairly similar effects on the above-mentioned parameters. However, different results were obtained when the effect of PFOA was assessed on LPO and complex II activity. Conclusions: Due to the fact that PFOA had toxic effects on the mitochondria isolated, it could be suggested that mitochondrial toxicity could be a plausible mechanism for the toxic effects of this fluorochemical on liver and brain function.