Biochemical and structural characterization of chlorhexidine as an ATP-assisted inhibitor against type 1 methionyl-tRNA synthetase from Gram-positive bacteria.

Methionyl-tRNA synthetase (MetRS) catalyzes the attachment of l-methionine (l-Met) to tRNAMet to generate methionyl-tRNAMet, an essential substrate for protein translation within ribosome. Owing to its indispensable biological function and the structural discrepancies with human counterpart, bacterial MetRS is considered an ideal target for developing antibacterials. Herein, chlorhexidine (CHX) was identified as a potent binder of Staphylococcus aureus MetRS (SaMetRS) through an ATP-aided affinity screening. The co-crystal structure showed that CHX simultaneously occupies the enlarged l-Met pocket (EMP) and the auxiliary pocket (AP) of SaMetRS with its two chlorophenyl groups, while its central hexyl linker swings upwards to interact with some conserved hydrophobic residues. ATP adopts alternative conformations in the active site cavity, and forms ionic bonds and water-mediated hydrogen bonds with CHX. Consistent with this synergistic binding mode, ATP concentration-dependently enhanced the binding affinity of CHX to SaMetRS from 10.2 µM (no ATP) to 0.45 µM (1 mM ATP). While it selectively inhibited two representative type 1 MetRSs from S. aureus and Enterococcus faecalis, CHX did not show significant interactions with three tested type 2 MetRSs, including human cytoplasmic MetRS, in the enzyme inhibition and biophysical binding assays, probably due to the conformational differences between two types of MetRSs at their EMP and AP. Our findings on CHX may inspire the design of MetRS-directed antimicrobials in future.

An asymmetric structure of bacterial TrpRS supports the half-of-the-sites catalytic mechanism and facilitates antimicrobial screening.

Tryptophanyl-tRNA synthetase (TrpRS) links tryptophan to tRNATrp, thereby playing an indispensable role in protein translation. Unlike most class I aminoacyl-tRNA synthetases (AARSs), TrpRS functions as a homodimer. Herein, we captured an 'open-closed' asymmetric structure of Escherichia coli TrpRS (EcTrpRS) with one active site occupied by a copurified intermediate product and the other remaining empty, providing structural evidence for the long-discussed half-of-the-sites reactivity of bacterial TrpRS. In contrast to its human counterpart, bacterial TrpRS may rely on this asymmetric conformation to functionally bind with substrate tRNA. As this asymmetric conformation is probably a dominant form of TrpRS purified from bacterial cells, we performed fragment screening against asymmetric EcTrpRS to support antibacterial discovery. Nineteen fragment hits were identified, and 8 of them were successfully cocrystallized with EcTrpRS. While a fragment named niraparib bound to the L-Trp binding site of the 'open' subunit, the other 7 fragments all bound to an unprecedented pocket at the interface between two TrpRS subunits. Binding of these fragments relies on residues specific to bacterial TrpRS, avoiding undesired interactions with human TrpRS. These findings improve our understanding of the catalytic mechanism of this important enzyme and will also facilitate the discovery of bacterial TrpRS inhibitors with therapeutic potential.

Discovery, optimization and evaluation of isothiazolo[5,4-b]pyridine derivatives as RIPK1 inhibitors with potent in vivo anti-SIRS activity.

Receptor-interacting protein kinase-1 (RIPK1) is involved in the necroptosis pathway, which regulates inflammatory signaling and cell death in a variety of diseases, including inflammatory and neurodegenerative disorders. We identified a novel hit compound 36 by a cell-based screening assay (anti-necroptosis EC50 = 58 nM). Starting from compound 36, we designed a series of scaffolds to improve anti-necroptosis activity, physicochemical properties and metabolic stability. The isothiazolo[5,4-b]pyridine backbone proved to be a promising scaffold which provided a number of potent necroptosis inhibitors. Compound 56, for example, effectively blocked necroptosis in both human and mouse cells (EC50 = 1-5 nM). A binding assay showed that compound 56 potently binds to RIPK1 (Kd = 13 nM), but not RIPK3 (Kd > 10,000 nM). Kinase functional assay (ADP-Glo) confirmed that compound 56 inhibits RIPK1 phosphorylation with an IC50 at 5.8 nM. Importantly, compound 56 displayed excellent cross-species liver microsomal metabolic stability (t1/2 > 90 min). Furthermore, compound 56 exhibited favorable in vitro safety profiles in hERG and CYP assays. Finally, pre-treatment with 56 significantly reduced hypothermia and lethal shock in the systemic inflammatory response syndrome mice model. Taken together, compound 56 represented a promising prototype for the development of therapeutic agent to treat inflammation-related diseases.

Ring closure strategy leads to potent RIPK3 inhibitors.

Necroptosis is a form of regulated necrotic cell death that is independent of caspases. Receptor-interacting protein kinase 3 (RIPK3) has been identified as a key regulator for necroptosis, and has been proposed as a potential therapeutic target for the treatment of diseases associated with necroptosis. In this report, we describe the design, synthesis, and evaluation of a series of novel RIPK3 inhibitors. The lead compound 38 exhibited potent activity (EC50 = 0.42 µM) in blocking TNFα, Smac mimetic and z-VAD (TSZ) induced cell death in HT-29 cells. Mechanistic studies showed that compound 38 bound to RIPK3 with high affinity (Kd = 7.1 nM), and inhibited RIPK3 kinase activity in a ADP-Glo functional assay. In addition, compound 38 displayed good selectivity over another necroptosis regulator RIPK1 (Kd = 6000 nM). Furthermore, compound 38 demonstrated excellent in vitro safety profiles with minimal inhibition of CYP isozymes and hERG potassium channel. Lastly, compound 38 efficiently blocked hypothermia and death in mice in the TNFα-induced systemic inflammatory response syndrome model.

Discovery of a Potent RIPK3 Inhibitor for the Amelioration of Necroptosis-Associated Inflammatory Injury.

Necroptosis is a form of regulated necrosis that requires the activation of receptor-interacting kinase 3 (RIPK3 or RIP3) and its phosphorylation of the substrate MLKL (mixed lineage kinase domain-like protein). Necroptosis has emerged as important cell death involved in the pathogenesis of various diseases including inflammatory diseases, degenerative diseases, and cancer. Here, we discovered a small molecule Zharp-99 as a potent inhibitor of necroptosis through blocking the kinase activity of RIPK3. Zharp-99 efficiently blocks necroptosis induced by ligands of the death receptor and Toll-like receptor as well as viral infection in human, rat and mouse cells. Zharp-99 strongly inhibits cellular activation of RIPK3, and MLKL upon necroptosis stimuli. Zharp-99 directly blocks the kinase activity of RIPK3 without affecting RIPK1 kinase activity at the tested concentration. Importantly, Zharp-99 exerts effective protection against TNF-α induced systemic inflammatory response syndrome in the mouse model. Zharp-99 displays favorable in vitro safety profiles and in vivo pharmacokinetic parameters. Thus, our study demonstrates Zharp-99 as a potent inhibitor of RIPK3 kinase and also highlights its potential for further development of new approaches for treating necroptosis-associated inflammatory disorders.

Design, synthesis, and evaluation of pyrrolidine based CXCR4 antagonists with in vivo anti-tumor metastatic activity.

The chemokine receptor CXCR4 has been proposed as a drug target based on its important functions in HIV infection, inflammation/autoimmune diseases and cancer metastasis. Herein we report the design, synthesis and evaluation of novel CXCR4 antagonists based on a pyrrolidine scaffold. The structural exploration/optimization identified numerous potent CXCR4 antagonists, represented by compound 46, which displayed potent binding affinity to CXCR4 receptor (IC50 = 79 nM competitively displacing fluorescent 12G5 antibody) and inhibited CXCL12 induced cytosolic calcium flux (IC50 = 0.25 nM). Moreover, in a transwell invasion assay, compound 46 significantly mitigated CXCL12/CXCR4 mediated cell migration. Compound 46 exhibited good physicochemical properties (MW 367, logD7.4 1.12, pKa 8.2) and excellent in vitro safety profiles (e.g., hERG patch clamp IC50 > 30 µM and minimal CYP isozyme inhibition). Importantly, 46 displayed much improved metabolic stability in human and rat liver microsomes. Lastly, 46 demonstrated marked efficacy in a cancer metastasis model in mice. These results strongly support 46 as a prototypical lead for the development of promising CXCR4 antagonists as clinical candidates.

Design, synthesis, and evaluation of novel CXCR4 antagonists based on an aminoquinoline template.

The chemokine receptor CXCR4 has been explored as a drug target due to its involvement in pathological conditions such as HIV infection and cancer metastasis. Here we report the structure-activity relationship study of novel CXCR4 antagonists based on an aminoquinoline template. This template is devoid of the chiral center in the classical tetrahydroquinoline (THQ) ring moiety and therefore can be easily synthesized. A number of potent CXCR4 antagonists were identified, exemplified by compound 3, which demonstrated excellent binding affinity with CXCR4 receptor (IC50 = 57 nM) and inhibited CXCL12 induced cytosolic calcium increase (IC50 = 0.24 nM). Furthermore, compound 3 potently inhibited CXLC12/CXCR4 mediated cell migration in a transwell invasion assay. The simplified synthetic approach combined with good physicochemical properties (e.g. MW 362, clogP 2.1, PSA 48, pKa 7.0 for compound 3) demonstrate the potential of this aminoquinoline template as a novel scaffold to develop CXCR4 antagonists.