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Monoamine Oxidase Reporters 1 and 2 (MR1 and MR2) are substrate-tethered activity probes that utilize a tandem amine oxidation/beta-elimination mechanism to detect MAO chemistry in vitro and in living cells directly and specifically without the need for additional enzymes or other activating reagents.

The development of low μM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K i = 1.4 ± 0.3 μM) was found to be selective when tested against a panel of human tyrosine and dual-specificity phosphatases (11-fold vs the highly homologous HCPtpA, and >70-fold vs all others tested). Modeling the inhibitor-PtpA complexes explained the structure-activity relationships observed in vitro and revealed further possibilities for compound development. KeywordsMycobacterium tuberculosis; Phosphatase; Inhibitor; PtpA Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Out of over 13 million active cases each year, TB causes nearly 2 million deaths. 1 Current treatment of drug-sensitive strains requires 6-9 months to fully eradicate the infection. New Mtb drugs that act on novel targets are needed to shorten treatment and address the emergence of antibiotic resistance.Mtb encodes two protein tyrosine phosphatases (PTPs), PtpA and PtpB, that are promising new targets for TB drug development. 2 These PTPs are secreted by Mtb 3 into the cytosol of infected macrophages, obviating the need for inhibitors to enter bacterial cells. 4 Although genetic deletion of PtpA or PtpB does not affect Mtb growth in culture, 4,5 these deletions severely attenuate growth in sensitive infected macrophages. 4 These data suggest that the Mtb PTPs act on macrophage signaling pathways to promote Mtb survival in the infected host. Although not classical drug targets because they are not essential in vitro, targeting the secreted PTPs in the host macrophage circumvents two central resistance mechanisms of Mtb; that is, poor drug permeability due to the Mtb cell wall, 6 and pump-mediated drug efflux. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptWe previously reported the development of low-molecular weight inhibitors of PtpB 8 using a substrate-based, fragment identification and optimization approach termed Substrate Activity Screening (SAS). 9 Here, we applied the same method to PtpA to prepare and evaluate a library of inhibitors selective for Mtb PtpA. These studies identified low-micromolar PtpA inhibitors with selectivity versus a panel of human phosphatases. Modeling our compounds bound in the active site of PtpA explained the observed structure-activity relationships (SAR) and highlighted further possibilities for compound development.A library of O-aryl phosphate substrate fragments was previously developed to target PtpB. 8 Using this library, we identified compounds for further optimization towards PtpA. Due to the ease of synthetic diversification of aryl difluoromethylphosphonic acid (DFMP) inhibitors, we varied DFMP analogs to establish SAR for PtpA inhibition. Although DFMP inhibitors have traditionally exhibited poor cell permeability due to the dianionic nature of this pharmacophore, DFMP inhibitors of the human phosphatase PTP1B, an enzyme involved in insulin signaling, have rece...

The design and synthesis of new inhibitor analogues for the Mycobacterium tuberculosis (Mtb) phosphatase PtpB is described. Analogues were synthesized by incorporation of two common and effective phosphate mimetics, the isothiazolidinone (IZD) and the difluoromethylphosphonic acid (DFMP). The basic scaffold of the inhibitor was identified from structure-activity relationships established for a previously published isoxazole inhibitor, while the phosphate mimetics were chosen based on their proven cell permeability and activity when incorporated into previously reported inhibitors for the phosphatase PTP1B. The inhibitory activity of each compound was evaluated, and each was found to have low or submicromolar affinity for PtpB. (Mtb). Each year, nearly 2 million deaths occur out of over 13 million active cases of TB, 1 and current treatment of drug-sensitive strains requires 6-9 months to fully eradicate the infection. New Mtb drugs that act on novel targets are needed to shorten treatment time and address the emergence of antibiotic resistance. Mtb PtpB is a secreted virulence factor that functions within human macrophages, and chemical interference with PtpB function has been shown to reduce bacterial loads in an Mtb macrophage infection model. 2 PtpB is attractive as a therapeutic target due to its localization outside of the unusually thick mycobacterial cell wall, which is difficult to penetrate and leads to long treatment times for tuberculosis (TB). Tuberculosis (TB) is a chronic, infectious disease caused by Mycobacterium tuberculosisWe previously reported the development of isoxazole carboxylic acid inhibitor 1 for PtpB, using the substrate activity screening (SAS) method, a novel fragment-based approach for the identification of phosphatase inhibitors (Figure 1). 3 Development of the isoxazole inhibitor revealed that ortho substituents are preferred for optimal PtpB binding, with the cyclohexyl group providing the most favorable enzyme interactions. In order to compare binding affinity and cell activity, we desired a route to analogueous inhibitors incorporating other commonly used phosphate mimetics. NIH-PA Author ManuscriptA variety of phosphate isosteres have been reported and could be introduced in place of the isoxazole,4 but many contain at least two acidic sites and lead to inhibitors with poor cellular permeability. 5 We therefore chose two phosphate mimetics that have been effectively used in inhibitors of PTP1B, a human phosphatase targeted for diabetes treatment, with good cell permeability and activity, namely the isothiazolidinone (IZD) and difluoromethylphosphonic acid (DFMP) mimetics ( Figure 2). The monoanionic IZD 6 phosphate mimetic was developed by Incyte through structure-based design, 6g while the DFMP pharmacophore is a commonly used phosphate mimetic that has been investigated extensively in the literature. 7 Despite being dianionic, the DFMP isostere has recently been shown by Merck to be cell permeable and orally bioavailable in animals when incorporated into nonpeptid...

Peptides containing N-alkylaminooxy amino acids were chemoselectively alkylated with allylic, benzylic, and alpha-carbonyl bromides, N-ethylmaleimide, and hexyl acrylate in mildly acidic aqueous/organic solutions. Alkylation at the aminooxy nitrogen proceeds in good yields with excellent to complete chemoselectivity in the presence of all common amino acids except cysteine. This reaction complements the selective glycosylation and acylation of N-alkylaminooxy groups and provides an avenue for the synthesis of peptide arrays comprising a wide variety of neoglycopeptides and neolipopeptides.