Aminocarboxylic acids related to aspergillomarasmine A (AMA) and ethylenediamine-N,N'-disuccinic acid (EDDS) are strong zinc-binders and inhibitors of the metallo-beta-lactamase NDM-1

Microbial Biotechnolog

Nicotinamide N-methyltransferase catalyses the N-methylation of the endogenous ß-carboline norharman: evidence for a novel detoxification pathway

Nicotinamide N-methyltransferase (NNMT) is responsible for the N-methylation of nicotinamide to 1-methylnicotinamide. Our recent studies have demonstrated that NNMT regulates cellular processes fundamental to the correct functioning and survival of the cell. It has been proposed that NNMT may possess β-carboline (BC) N-methyltransferase activity, endogenously and exogenously produced pyridine-containing compounds which, when N-methylated, are potent inhibitors of Complex I and have been proposed to have a role in the pathogenesis of Parkinson's disease. We have investigated the ability of recombinant NNMT to N-methylate norharman (NH) to 2-N-methylnorharman (MeNH). In addition, we have investigated the toxicity of the BC NH, its precursor 1,2,3,4-tetrahydronorharman (THNH) and its N-methylated metabolite MeNH, using our in vitro SH-SY5Y NNMT expression model. Recombinant NNMT demonstrated NH 2N-methyltransferase activity, with a Km of 90 ± 20 µM, a kcat of 3 × 10(-4) ± 2 × 10(-5) s(-1) and a specificity constant (kcat/Km) of 3 ± 1 s(-1) M(-1) THNH was the least toxic of all three compounds investigated, whereas NH demonstrated the greatest, with no difference observed in terms of cell viability and cell death between NNMT-expressing and non-expressing cells. In NNMT-expressing cells, MeNH increased cell viability and cellular ATP concentration in a dose-dependent manner after 72 and 120 h incubation, an effect that was not observed after 24 h incubation or in non-NNNT-expressing cells at any time point. Taken together, these results suggest that NNMT may be a detoxification pathway for BCs such as NH

Inhibitors of nicotinamide:N -methyltransferase designed to mimic the methylation reaction transition state

Nicotinamide N-methyltransferase (NNMT) is an enzyme that catalyses the methylation of nicotinamide to form N'-methylnicotinamide. Both NNMT and its methylated product have recently been linked to a variety of diseases, suggesting a role for the enzyme as a therapeutic target beyond its previously ascribed metabolic function in detoxification. We here describe the systematic development of NNMT inhibitors derived from the structures of the substrates involved in the methylation reaction. By covalently linking fragments of the NNMT substrates a diverse library of bisubstrate-like compounds was prepared. The ability of these compounds to inhibit NNMT was evaluated providing valuable insights into the structural tolerances of the enzyme active site. These studies led to the identification of new NNMT inhibitors that mimic the transition state of the methylation reaction and inhibit the enzyme with activity on par with established methyltransferase inhibitors

MYC is a clinically significant driver of mTOR inhibitor resistance in breast cancer

Targeting the PI3K–AKT–mTOR pathway is a promising therapeutic strategy for breast cancer treatment. However, low response rates and development of resistance to PI3K–AKT–mTOR inhibitors remain major clinical challenges. Here, we show that MYC activation drives resistance to mTOR inhibitors (mTORi) in breast cancer. Multiomic profiling of mouse invasive lobular carcinoma (ILC) tumors revealed recurrent Myc amplifications in tumors that acquired resistance to the mTORi AZD8055. MYC activation was associated with biological processes linked to mTORi response and counteracted mTORi-induced translation inhibition by promoting translation of ribosomal proteins. In vitro and in vivo induction of MYC conferred mTORi resistance in mouse and human breast cancer models. Conversely, AZD8055-resistant ILC cells depended on MYC, as demonstrated by the synergistic effects of mTORi and MYCi combination treatment. Notably, MYC status was significantly associated with poor response to everolimus therapy in metastatic breast cancer patients. Thus, MYC is a clinically relevant driver of mTORi resistance that may stratify breast cancer patients for mTOR-targeted therapies

A Rapid and Efficient Assay for the Characterization of Substrates and Inhibitors of Nicotinamide N-Methyltransferase

Nicotinamide <i>N</i>-methyltransferase (NNMT) is one of the most abundant small molecule methyltransferases in the human body and is primarily responsible for the N-methylation of the nicotinamide (vitamin B3). Employing the cofactor <i>S</i>-adenosyl-l-methionine, NNMT transfers a methyl group to the pyridine nitrogen of nicotinamide to generate <i>N</i>-methylnicotinamide. Interestingly, NNMT is also able to N-methylate a variety of other pyridine-containing small molecules, suggesting a secondary role for the enzyme in the detoxification of xenobiotics. A number of recent studies have also revealed links between NNMT overexpression and a variety of diseases, including multiple cancers, Parkinson’s disease, diabetes, and obesity. To facilitate further study of both the substrate scope and potential for inhibitor development, we here describe the development of a new NNMT activity assay. The assay makes use of ultra-high-performance hydrophilic interaction chromatography, allowing for rapid separation of the reaction products, coupled with quadrupole time-of-flight mass spectrometric detection, providing for enhanced sensitivity and enabling high-throughput sample analysis. We successfully demonstrated the general applicability of the method by performing kinetic analyses of NNMT-mediated methylation for a range of pyridine-based substrates. These findings also provide new insight into the diversity of substrate recognition by NNMT in a quantitative manner. In addition, we further established the suitability of the assay for the identification and characterization of small molecule inhibitors of NNMT. To do so, we investigated the inhibition of NNMT by the nonspecific methyltransferase inhibitors sinefungin and <i>S</i>-adenosyl-l-homocysteine, revealing IC₅₀ values in the low micromolar range. The results of these inhibition studies are particularly noteworthy as they will permit future efforts toward the development of new NNMT-specific 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 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

Macrocyclic Peptides as Allosteric Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide using S-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In search of structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC50 values as low as 229 nM. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.</p

Novel Cephalosporin Conjugates Display Potent and Selective Inhibition of IMP Type Metallo-β-Lactamases

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