Discovery of Novel UDP-N-Acetylglucosamine Acyltransferase (LpxA) Inhibitors with Activity against Pseudomonas aeruginosa.

This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 µg/mL). Lack of activity against E. coli was maintained (IC50 > 20 µM and MIC > 128 µg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.

Tetrahydroindazoles as Interleukin-2 Inducible T-Cell Kinase Inhibitors. Part II. Second-Generation Analogues with Enhanced Potency, Selectivity, and Pharmacodynamic Modulation in Vivo.

The medicinal chemistry community has directed considerable efforts toward the discovery of selective inhibitors of interleukin-2 inducible T-cell kinase (ITK), given its role in T-cell signaling downstream of the T-cell receptor (TCR) and the implications of this target for inflammatory disorders such as asthma. We have previously disclosed a structure- and property-guided lead optimization effort which resulted in the discovery of a new series of tetrahydroindazole-containing selective ITK inhibitors. Herein we disclose further optimization of this series that resulted in further potency improvements, reduced off-target receptor binding liabilities, and reduced cytotoxicity. Specifically, we have identified a correlation between the basicity of solubilizing elements in the ITK inhibitors and off-target antiproliferative effects, which was exploited to reduce cytotoxicity while maintaining kinase selectivity. Optimized analogues were shown to reduce IL-2 and IL-13 production in vivo following oral or intraperitoneal dosing in mice.

Property- and structure-guided discovery of a tetrahydroindazole series of interleukin-2 inducible T-cell kinase inhibitors.

Interleukin-2 inducible T-cell kinase (ITK), a member of the Tec family of tyrosine kinases, plays a major role in T-cell signaling downstream of the T-cell receptor (TCR), and considerable efforts have been directed toward discovery of ITK-selective inhibitors as potential treatments of inflammatory disorders such as asthma. Using a previously disclosed indazole series of inhibitors as a starting point, and using X-ray crystallography and solubility forecast index (SFI) as guides, we evolved a series of tetrahydroindazole inhibitors with improved potency, selectivity, and pharmaceutical properties. Highlights include identification of a selectivity pocket above the ligand plane, and identification of appropriate lipophilic substituents to occupy this space. This effort culminated in identification of a potent and selective ITK inhibitor (GNE-9822) with good ADME properties in preclinical species.

Structure-based design and synthesis of potent benzothiazole inhibitors of interleukin-2 inducible T cell kinase (ITK).

Inhibition of the non-receptor tyrosine kinase ITK, a component of the T-cell receptor signalling cascade, may represent a novel treatment for allergic asthma. Here we report the structure-based optimization of a series of benzothiazole amides that demonstrate sub-nanomolar inhibitory potency against ITK with good cellular activity and kinase selectivity. We also elucidate the binding mode of these inhibitors by solving the X-ray crystal structures of several inhibitor-ITK complexes.

Impact of epimerization domains on the intermodular transfer of enzyme-bound intermediates in nonribosomal peptide synthesis.

Assembly of bioactive natural compounds through the action of nonribosomal peptide synthetases (NRPSs) relies on the specific interplay of modules and domains along these multiple mega-enzymes. As the C termini of several bacterial NRPSs often harbor epimerization (E) domains that generate D-amino acids, these seem to facilitate the ordered intermolecular enzymatic interaction and the directed transfer of intermediates. To elucidate this bifunctional role, E domains in recombinant bimodular proteins derived from the tyrocidine synthetase B were investigated. By utilizing sequent tryptic proteolysis and HPLC Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), we could directly interrogate and determine the formation of intermediates attached to the TycB(3)-PCP domain of wild-type TycB(2-3) and to the E domain exchange enzyme TycB(2-3)-ATCAT/E(tycA). In addition, the two proteins and a version of TycB(2-3) fused to the communication-mediating (COM) domain of TycA were applied in product formation assays with TycB(1) to corroborate E domain impact on intermodular NRPS interaction. Significant functional differences between the C-terminal aminoacyl- and peptidyl-E domains were observed in terms of in trans interaction and misinitiation. E domains originating from elongation modules (peptidyl-E domains) seem to be optimized for regulation of the progression of peptide bond formation, epimerization, and intermediate transfer to the downstream module, whereas E domains of initiation modules (aminoacyl-E domains) impair upstream condensation and cause misinitiation. The selection of E domains is therefore decisive for successful application in biocombinatorial engineering of nonribosomal peptides.

Utility of epimerization domains for the redesign of nonribosomal peptide synthetases.

Many pharmacologically important agents are assembled on multimodular nonribosomal peptide synthetases (NRPSs) whose modules comprise a set of core domains with all essential catalytic functions necessary for the incorporation and modification of one building block. Very often, d-amino acids are found in such products which, with few exceptions, are generated by the action of NRPS integrated epimerization (E) domains that alter the stereochemistry of the corresponding peptidyl carrier protein (PCP) bound l-intermediate. In this study we present a quantitative investigation of substrate specificity of four different E domains (two 'peptidyl-' and two 'aminoacyl-'E domains) derived from different NRPSs towards PCP bound peptides. The respective PCP-E bidomain apo-proteins (TycB(3)-, FenD(2)-, TycA- and GrsA-PCP-E) were primed with various peptidyl-CoA precursors by utilizing the promiscuous phosphopantetheinyl transferase Sfp. PCP bound peptidyl-S-Ppant epimerization products were chemically cleaved and analyzed for their l/d-ratios by LCMS. We were able to show that all four E domains tolerate a broad variety of peptidyl-S-Ppant-substrates as evaluated by k(obs) values and final l/d-product equilibria determined for each reaction. The two C-terminal amino acids of the substrate seem to be recognized by 'peptidyl-'E domains. Interestingly, the 'aminoacyl-'E domains GrsA- and TycA-E were also able to convert the elongated intermediates. All four E domains accepted an N-methylated precursor as well and epimerized this substrate with high efficiency. Finally, we could demonstrate that the condensation (C) domain of TycB(1) is also able to process peptidyl substrates transferred by TycA. In conclusion, these findings are of great impact on future engineering attempts.

Systematic and quantitative analysis of protein-protein recognition between nonribosomal peptide synthetases investigated in the tyrocidine biosynthetic template.

We present a systematic and quantitative study of the protein-protein recognition between the three tyrocidine synthetases TycA, TycB, and TycC investigated with two artificial in trans assay systems, which had been previously developed: the "DKP assay system" for the interaction of TycA with TycB and the "L/D-Phe-L-Asn assay system" for the interaction of TycB with TycC. TycA-A(Phe)TE and TycB(3)-A(Phe)TE, which are used as donor enzymes, both provide D-Phe-S-Ppant, so that no substrate specificities interfered with the quantification of protein-protein recognition. We tested all donor/acceptor enzyme combinations between the two artificial assay systems for product formation activities as well as two hybrid enzymes, where the E-domains of TycA and TycB(3) had been exchanged against each other. Furthermore, four donor/acceptor protein fusions were constructed on gene level, resulting in dimodular proteins. We were able to show that the E-domains mediate protein-protein recognition in trans. Product formation of the different donor assayed with the two acceptor enzymes TycB(1)-CA(Pro)T and TycC(1)-CA(Asn)T/Te in trans was only obtained if the donor enzyme harbored the cognate E-domain. Interestingly, all in cis fusions (dimodular proteins) were active, giving strong evidence that unnatural protein-protein interactions can be "forced" by fusion of the distinct enzymes. Finally, we were able to detect product formation in the "DKP system" with engineered hybrid proteins where the A-domain of TycA had been exchanged against the isoleucine-activating A-domain of BacA(1) and the valine-activating A-domain of TycC(4), respectively. All of these findings are of high relevance for future NRPS engineering approaches.