A Combined Approach Reveals a Regulatory Mechanism Coupling Src's Kinase Activity, Localization, and Phosphotransferase-Independent Functions.

Multiple layers of regulation modulate the activity and localization of protein kinases. However, many details of kinase regulation remain incompletely understood. Here, we apply saturation mutagenesis and a chemical genetic method for allosterically modulating kinase global conformation to Src kinase, providing insight into known regulatory mechanisms and revealing a previously undiscovered interaction between Src's SH4 and catalytic domains. Abrogation of this interaction increased phosphotransferase activity, promoted membrane association, and provoked phosphotransferase-independent alterations in cell morphology. Thus, Src's SH4 domain serves as an intramolecular regulator coupling catalytic activity, global conformation, and localization, as well as mediating a phosphotransferase-independent function. Sequence conservation suggests that the SH4 domain regulatory interaction exists in other Src-family kinases. Our combined approach's ability to reveal a regulatory mechanism in one of the best-studied kinases suggests that it could be applied broadly to provide insight into kinase structure, regulation, and function.

Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model.

BACKGROUND: Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. OBJECTIVES: To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. METHODS: Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. RESULTS: Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. CONCLUSIONS: The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays.

In Toxoplasma gondii, calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC50s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC50s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC50s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC50s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.

Active site profiling reveals coupling between domains in SRC-family kinases.

Protein kinases, key regulators of intracellular signal transduction, have emerged as an important class of drug targets. Chemical proteomic tools that facilitate the functional interrogation of protein kinase active sites are powerful reagents for studying the regulation of this large enzyme family and performing inhibitor selectivity screens. Here we describe a new crosslinking strategy that enables rapid and quantitative profiling of protein kinase active sites in lysates and live cells. Applying this methodology to the SRC-family kinases (SFKs) SRC and HCK led to the identification of a series of conformation-specific, ATP-competitive inhibitors that have a distinct preference for the autoinhibited forms of these kinases. Furthermore, we show that ligands that have this selectivity are able to modulate the ability of the regulatory domains of SRC and HCK to engage in intermolecular binding interactions. These studies provide insight into the regulation of this important family of tyrosine kinases.

The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites.

Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

To B or not to B: a question of resolution?

In choosing and refining any crystallographic structural model, there is tension between the desire to extract the most detailed information possible and the necessity to describe no more than what is justified by the observed data. A more complex model is not necessarily a better model. Thus, it is important to validate the choice of parameters as well as validating their refined values. One recurring task is to choose the best model for describing the displacement of each atom about its mean position. At atomic resolution one has the option of devoting six model parameters (a 'thermal ellipsoid') to describe the displacement of each atom. At medium resolution one typically devotes at most one model parameter per atom to describe the same thing (a 'B factor'). At very low resolution one cannot justify the use of even one parameter per atom. Furthermore, this aspect of the structure may be described better by an explicit model of bulk displacements, the most common of which is the translation/libration/screw (TLS) formalism, rather than by assigning some number of parameters to each atom individually. One can sidestep this choice between atomic displacement parameters and TLS descriptions by including both treatments in the same model, but this is not always statistically justifiable. The choice of which treatment is best for a particular structure refinement at a particular resolution can be guided by general considerations of the ratio of model parameters to the number of observations and by specific statistics such as the Hamilton R-factor ratio test.

Structure of the prolyl-tRNA synthetase from the eukaryotic pathogen Giardia lamblia.

The genome of the human intestinal parasite Giardia lamblia contains only a single aminoacyl-tRNA synthetase gene for each amino acid. The Giardia prolyl-tRNA synthetase gene product was originally misidentified as a dual-specificity Pro/Cys enzyme, in part owing to its unexpectedly high off-target activation of cysteine, but is now believed to be a normal representative of the class of archaeal/eukaryotic prolyl-tRNA synthetases. The 2.2 Å resolution crystal structure of the G. lamblia enzyme presented here is thus the first structure determination of a prolyl-tRNA synthetase from a eukaryote. The relative occupancies of substrate (proline) and product (prolyl-AMP) in the active site are consistent with half-of-the-sites reactivity, as is the observed biphasic thermal denaturation curve for the protein in the presence of proline and MgATP. However, no corresponding induced asymmetry is evident in the structure of the protein. No thermal stabilization is observed in the presence of cysteine and ATP. The implied low affinity for the off-target activation product cysteinyl-AMP suggests that translational fidelity in Giardia is aided by the rapid release of misactivated cysteine.

Affinity-based probes based on type II kinase inhibitors.

Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.

Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1.

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.

Selective inhibitors of methionyl-tRNA synthetase have potent activity against Trypanosoma brucei Infection in Mice.

Human African trypanosomiasis continues to be an important public health threat in extensive regions of sub-Saharan Africa. Treatment options for infected patients are unsatisfactory due to toxicity, difficult administration regimes, and poor efficacy of available drugs. The aminoacyl-tRNA synthetases were selected as attractive drug targets due to their essential roles in protein synthesis and cell survival. Comparative sequence analysis disclosed differences between the trypanosome and mammalian methionyl-tRNA synthetases (MetRSs) that suggested opportunities for selective inhibition using drug-like molecules. Experiments using RNA interference on the single MetRS of Trypanosoma brucei demonstrated that this gene product was essential for normal cell growth. Small molecules (diaryl diamines) similar to those shown to have potent activity on prokaryotic MetRS enzymes were synthesized and observed to have inhibitory activity on the T. brucei MetRS (50% inhibitory concentration, <50 nM) and on bloodstream forms of T. brucei cultures (50% effective concentration, as low as 4 nM). Twenty-one compounds had a close correlation between enzyme binding/inhibition and T. brucei growth inhibition, indicating that they were likely to be acting on the intended target. The compounds had minimal effects on mammalian cell growth at 20 µM, demonstrating a wide therapeutic index. The most potent compound was tested in the murine model of trypanosomiasis and demonstrated profound parasite suppression and delayed mortality. A homology model of the T. brucei MetRS based on other MetRS structures was used to model binding of the lead diaryl diamine compounds. Future studies will focus on improving the pharmacological properties of the MetRS inhibitors.

Screening a fragment cocktail library using ultrafiltration.

Ultrafiltration provides a generic method to discover ligands for protein drug targets with millimolar to micromolar K(d), the typical range of fragment-based drug discovery. This method was tailored to a 96-well format, and cocktails of fragment-sized molecules, with molecular masses between 150 and 300 Da, were screened against medical structural genomics target proteins. The validity of the method was confirmed through competitive binding assays in the presence of ligands known to bind the target proteins.

The structure of tryptophanyl-tRNA synthetase from Giardia lamblia reveals divergence from eukaryotic homologs.

The 2.1A crystal structure of tryptophanyl-tRNA synthetase (TrpRS) from the diplomonad Giardia lamblia reveals that the N-terminus of this class I aminoacyl-tRNA synthetase forms a 16-residue alpha-helix. This helix replaces a beta-hairpin that is required by human TrpRS for normal activity and has been inferred to play a similar role in all eukaryotic TrpRS. The primary sequences of TrpRS homologs from several basal eukaryotes including Giardia lack a set of three residues observed to stabilize interactions with this beta-hairpin in the human TrpRS. Thus the present structure suggests that the activation reaction mechanism of TrpRS from the basal eukaryote G. lamblia differs from that of higher eukaryotes. Furthermore, the protein as observed in the crystal forms an (alpha(2))(2) homotetramer. The canonical dimer interface observed in all previous structures of tryptophanyl-tRNA synthetases is maintained, but in addition each N-terminal alpha-helix reciprocally interlocks with the equivalent helix from a second dimer to form a dimer of dimers. Although we have no evidence for tetramer formation in vivo, modeling indicates that the crystallographically observed tetrameric structure would be compatible with the tRNA binding mode used by dimeric TrpRS and TyrRS.

Prediction of protein crystallization outcome using a hybrid method.

The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R(30), the ratio between a protein's DSF intensity at 30°C and at T(m). This hybrid model is shown to be more powerful than sequence-based prediction alone - and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories.

Toxoplasma gondii cathepsin L is the primary target of the invasion-inhibitory compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl.

The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 A resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.

Validation of crystallographic models containing TLS or other descriptions of anisotropy.

The use of TLS (translation/libration/screw) models to describe anisotropic displacement of atoms within a protein crystal structure has become increasingly common. These models may be used purely as an improved methodology for crystallographic refinement or as the basis for analyzing inter-domain and other large-scale motions implied by the crystal structure. In either case it is desirable to validate that the crystallographic model, including the TLS description of anisotropy, conforms to our best understanding of protein structures and their modes of flexibility. A set of validation tests has been implemented that can be integrated into ongoing crystallographic refinement or run afterwards to evaluate a previously refined structure. In either case validation can serve to increase confidence that the model is correct, to highlight aspects of the model that may be improved or to strengthen the evidence supporting specific modes of flexibility inferred from the refined TLS model. Automated validation checks have been added to the PARVATI and TLSMD web servers and incorporated into the CCP4i user interface.

Atomic resolution studies of carbonic anhydrase II.

Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9 A resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25 A) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01 A in some zinc-protein and zinc-ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available.

Nek8445, a protein kinase required for microtubule regulation and cytokinesis in Giardia lamblia.

Giardia has 198 Nek kinases whereas humans have only 11. Giardia has a complex microtubule cytoskeleton that includes eight flagella and several unique microtubule arrays that are utilized for parasite attachment and facilitation of rapid mitosis and cytokinesis. The need to regulate these structures may explain the parallel expansion of the number of Nek family kinases. Here we use live and fixed cell imaging to uncover the role of Nek8445 in regulating Giardia cell division. We demonstrate that Nek8445 localization is cell cycle regulated and this kinase has a role in regulating overall microtubule organization. Nek8445 depletion results in short flagella, aberrant ventral disk organization, loss of the funis, defective axoneme exit, and altered cell shape. The axoneme exit defect is specific to the caudal axonemes, which exit from the posterior of the cell, and this defect correlates with rounding of the cell posterior and loss of the funis. Our findings implicate a role for the funis in establishing Giardia's cell shape and guiding axoneme docking. On a broader scale our results support the emerging view that Nek family kinases have a general role in regulating microtubule organization.

Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies.

Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 A. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.

HingeMaster: normal mode hinge prediction approach and integration of complementary predictors.

Protein motion is often the link between structure and function and a substantial fraction of proteins move through a domain hinge bending mechanism. Predicting the location of the hinge from a single structure is thus a logical first step towards predicting motion. Here, we describe ways to predict the hinge location by grouping residues with correlated normal-mode motions. We benchmarked our normal-mode based predictor against a gold standard set of carefully annotated hinge locations taken from the Database of Macromolecular Motions. We then compared it with three existing structure-based hinge predictors (TLSMD, StoneHinge, and FlexOracle), plus HingeSeq, a sequence-based hinge predictor. Each of these methods predicts hinges using very different sources of information-normal modes, experimental thermal factors, bond constraint networks, energetics, and sequence, respectively. Thus it is logical that using these algorithms together would improve predictions. We integrated all the methods into a combined predictor using a weighted voting scheme. Finally, we encapsulated all our results in a web tool which can be used to run all the predictors on submitted proteins and visualize the results.