High Target Homology Does Not Guarantee Inhibition: Aminothiazoles Emerge as Inhibitors of Plasmodium falciparum.

In this study, we identified three novel compound classes with potent activity against Plasmodium falciparum, the most dangerous human malarial parasite. Resistance of this pathogen to known drugs is increasing, and compounds with different modes of action are urgently needed. One promising drug target is the enzyme 1-deoxy-d-xylulose-5-phosphate synthase (DXPS) of the methylerythritol 4-phosphate (MEP) pathway for which we have previously identified three active compound classes against Mycobacterium tuberculosis. The close structural similarities of the active sites of the DXPS enzymes of P. falciparum and M. tuberculosis prompted investigation of their antiparasitic action, all classes display good cell-based activity. Through structure-activity relationship studies, we increased their antimalarial potency and two classes also show good metabolic stability and low toxicity against human liver cells. The most active compound 1 inhibits the growth of blood-stage P. falciparum with an IC50 of 600 nM. The results from three different methods for target validation of compound 1 suggest no engagement of DXPS. All inhibitor classes are active against chloroquine-resistant strains, confirming a new mode of action that has to be further investigated.

Methods for Structure Determination of SH2 Domain-Phosphopeptide Complexes by NMR.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to solve the structure of biomolecular complexes at atomic resolution in solution. Small proteins such as Src-homology 2 (SH2) domains have fast tumbling rates and long-lived NMR signals, making them particularly suited to be studied by standard NMR methods. SH2 domains are modular proteins whose function is the recognition of sequences containing phosphotyrosines. In this chapter, we describe the application of NMR to assess the interaction between SH2 domains and phosphopeptides and determine the structure of the resulting complexes.

NMR Methods to Study the Dynamics of SH2 Domain-Phosphopeptide Complexes.

Nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying the dynamics of biological macromolecules in solution. By exploiting the intricate interplay between the effects of protein motion (both overall rotational diffusion and internal mobility) and nuclear spin relaxation, NMR allows molecular motion to be probed at atomic resolution over a wide range of timescales, including picosecond (bond vibrations and methyl-group rotations), nanosecond (loop motions and rotational diffusion), and microsecond-millisecond (ligand binding, allostery). In this chapter, we describe different NMR pulse schemes (R1, R1ρ, heteronuclear NOE, and CPMG relaxation dispersion) to characterize the dynamics of SH2 domains. As an example, we use the N-SH2 domain of protein tyrosine phosphatase SHP2 in complex with two phosphopeptides derived from immune checkpoint receptor PD-1 (ITIM and ITSM).

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation.

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman's Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells. Here, we show that a phosphorylated Y481EEVL motif in pK15 preferentially binds into the PLCγ1 C-terminal SH2 domain (cSH2), which is involved in conformational changes occurring during the activation of PLCγ1 by receptor tyrosine kinases. We determined the crystal structure of a pK15 12mer peptide containing the phosphorylated pK15 Y481EEVL motif in complex with a shortened PLCγ1 tandem SH2 (tSH2) domain. This structure demonstrates that the pK15 peptide binds to the PLCγ1 cSH2 domain in a position that is normally occupied by the linker region connecting the PLCγ1 cSH2 and SH3 domains. We also show that longer pK15 peptides containing the phosphorylated pK15 Y481EEVL motif can increase the Src-mediated phosphorylation of the PLCγ1 tSH2 region in vitro. This pK15-induced increase in Src-mediated phosphorylation of PLCγ1 can be inhibited with the small pK15-derived peptide which occupies the PLCγ1 cSH2 domain. Our findings thus suggest that pK15 may act as a scaffold protein to promote PLCγ1 activation in a manner similar to the cellular scaffold protein SLP-76, which has been shown to promote PLCγ1 activation in the context of T-cell receptor signaling. Reminiscent of its positional homologue in Epstein-Barr Virus, LMP2A, pK15 may therefore mimic aspects of antigen-receptor signaling. Our findings also suggest that it may be possible to inhibit the recruitment and activation of PLCγ1 pharmacologically.

Phosphotyrosine couples peptide binding and SHP2 activation via a dynamic allosteric network.

SHP2 is a ubiquitous protein tyrosine phosphatase, whose activity is regulated by phosphotyrosine (pY)-containing peptides generated in response to extracellular stimuli. Its crystal structure reveals a closed, auto-inhibited conformation in which the N-terminal Src homology 2 (N-SH2) domain occludes the catalytic site of the phosphatase (PTP) domain. High-affinity mono-phosphorylated peptides promote catalytic activity by binding to N-SH2 and disrupting the interaction with the PTP. The mechanism behind this process is not entirely clear, especially because N-SH2 is incapable of accommodating complete peptide binding when SHP2 is in the auto-inhibited state. Here, we show that pY performs an essential role in this process; in addition to its contribution to overall peptide-binding energy, pY-recognition leads to enhanced dynamics of the N-SH2 EF and BG loops via an allosteric communication network, which destabilizes the N-SH2-PTP interaction surface and simultaneously generates a fully accessible binding pocket for the C-terminal half of the phosphopeptide. Subsequently, full binding of the phosphopeptide is associated with the stabilization of activated SHP2. We demonstrate that this allosteric network exists only in N-SH2, which is directly involved in the regulation of SHP2 activity, while the C-terminal SH2 domain (C-SH2) functions primarily to recruit high-affinity bidentate phosphopeptides.