Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

Abstract

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...

Mycobacterium tuberculosis Serine/Threonine Protein Kinases

“…Chiradia et al identified synthetic chalcones as inhi bitors of PtpA, and in follow-up work elucidated structure-activity relationships and demonstrated inhibition by one compound of M. bovis BCG in human macrophages but not in axenic culture ( 108 – 110 ). A group that had previously identified PtpB inhibitors using a fragment-based approach applied this method to identify inhibitors of PtpA ( 111 ). The most potent of these ( K i = 1.4 μM) was also selective relative to human Tyr and dual-specificity kinases.…”

Dynamic Substrate Enhancement for the Identification of Specific, Second‐Site‐Binding Fragments Targeting a Set of Protein Tyrosine Phosphatases

“…Compounds 6 and 7 can compete well with the potency and selectivity of earlier MptpA inhibitors. [25][26][27] …”

Finding the Smoking Gun: Protein Tyrosine Phosphatases as Tools and Targets of Unicellular Microorganisms and Viruses

“…Although the deletion of any of the two PTPs does not affect the pathogen growth in cell culture, the identical deletions severely attenuate growth in infected host macrophages [90,94]. As the PTPs are not essential for the growth of the pathogen itself, but are essential for the modulation of host signaling pathways to allow the persistence of the pathogen in the infected host, the pharmacotherapeutic targeting of PtpA and PtpB must focus on the drug permeability in the host macrophage, but not in the M. tuberculosis (carrying the cell wall and efficient pump-mediated drug efflux systems [95]). …”

“…(3)) (K i = 1.4 µM against PtpA) is selective when tested against a panel of human PTPs and DUSPs (>70-fold selective over human PTP1B, TC-PTP, VHR, CD45, and LAR; 11-fold selective over human LMW-PTP), and does not inhibit M. tuberculosis PtpB (>100-fold selectivity). Molecular modeling highlighted the importance of pi-stacking with Trp48 of PtpA for high-affinity binding [95]. Regarding PtpB inhibition, isoxazole carboxylic acid isostere 433 Da analogue (13) has K i 220 nM against PtpB (>100-fold selectivity over M. tuberculosis PtpA, as well as over human VHR and TC-PTP, 98-fold selectivity over human LAR, 35-fold selectivity over human CD45) [97].…”

“…The low-molecular inhibitors of PtpA [95] and PtpB [97] were reported, all of them include the O-aryl phosphate substrate fragments [97]. The most potent of these benzanilide-scaffoldbased inhibitors (12) (Fig.…”