Allosteric activation mechanism of the Mycobacterium tuberculosis receptor Ser/Thr protein kinase, PknB

The essential Mycobacterium tuberculosis Ser/Thr protein kinase (STPK), PknB, plays a key role in regulating growth and division, but the structural basis of activation has not been defined. Here, we provide biochemical and structural evidence that dimerization through the kinase-domain (KD) N-lobe activates PknB by an allosteric mechanism. Promoting KD pairing using a small-molecule dimerizer stimulates the unphosphorylated kinase, and substitutions that disrupt N-lobe pairing decrease phosphorylation activity in vitro and in vivo. Multiple crystal structures of two monomeric PknB KD mutants in complex with nucleotide reveal diverse inactive conformations that contain large active-site distortions that propagate > 30 ? from the mutation site. These results define flexible, inactive structures of a monomeric bacterial receptor KD and show how "back-to-back" N-lobe dimerization stabilizes the active KD conformation. This general mechanism of bacterial receptor STPK activation affords insights into the regulation of homologous eukaryotic kinases that form structurally similar dimers.     

New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis

Background

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.

Methodology/Principal Findings

To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the ‘hits’ for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.

Conclusions/Significance

This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.     

Structural insight into the Mycobacterium tuberculosis Rv0020c protein and its interaction with the PknB kinase

The protein Rv0020c from Mycobacterium tuberculosis, also called FhaA, is one of the major substrates of the essential Ser/Thr protein kinase (STPK) PknB. The protein is composed of three domains and is phosphorylated on a unique site in its N terminus. We solved the solution structure of both N- and C-terminal domains and demonstrated that the approximately 300 amino acids of the intermediate domain are not folded. We present evidence that the FHA, a phosphospecific binding domain, of Rv0020c does not interact with the phosphorylated catalytic domains of PknB, but with the phosphorylated juxtamembrane domain that links the catalytic domain to the mycobacterial membrane. We also demonstrated that the degree and the pattern of phosphorylation of this juxtamembrane domain modulates the affinity of the substrate (Rv0020c) toward its kinase (PknB).     

The extracytoplasmic domain of the Mycobacterium tuberculosis Ser/Thr kinase PknB binds specific muropeptides and is required for PknB localization

The Mycobacterium tuberculosis Ser/Thr kinase PknB has been implicated in the regulation of cell growth and morphology in this organism. The extracytoplasmic domain of this membrane protein comprises four penicillin binding protein and Ser/Thr kinase associated (PASTA) domains, which are predicted to bind stem peptides of peptidoglycan. Using a comprehensive library of synthetic muropeptides, we demonstrate that the extracytoplasmic domain of PknB binds muropeptides in a manner dependent on the presence of specific amino acids at the second and third positions of the stem peptide, and on the presence of the sugar moiety N-acetylmuramic acid linked to the peptide. We further show that PknB localizes strongly to the mid-cell and also to the cell poles, and that the extracytoplasmic domain is required for PknB localization. In contrast to strong growth stimulation by conditioned medium, we observe no growth stimulation of M. tuberculosis by a synthetic muropeptide with high affinity for the PknB PASTAs. We do find a moderate effect of a high affinity peptide on resuscitation of dormant cells. While the PASTA domains of PknB may play a role in stimulating growth by binding exogenous peptidoglycan fragments, our data indicate that a major function of these domains is for proper PknB localization, likely through binding of peptidoglycan fragments produced locally at the mid-cell and the cell poles. These data suggest a model in which PknB is targeted to the sites of peptidoglycan turnover to regulate cell growth and cell division.     

Auto‐activation mechanism of the Mycobacterium tuberculosis PknB receptor Ser/Thr kinase

Many Ser/Thr protein kinases are activated by autophosphorylation, but the mechanism of this process has not been defined. We determined the crystal structure of a mutant of the Ser/Thr kinase domain (KD) of the mycobacterial sensor kinase PknB in complex with an ATP competitive inhibitor and discovered features consistent with an activation complex. The complex formed an asymmetric dimer, with the G helix and the ordered activation loop of one KD in contact with the G helix of the other. The activation loop of this putative ‘substrate’ KD was disordered, with the ends positioned at the entrance to the partner KD active site. Single amino‐acid substitutions in the G‐helix interface reduced activation‐loop phosphorylation, and multiple replacements abolished KD phosphorylation and kinase activation. Phosphorylation of an inactive mutant KD was reduced by G‐helix substitutions in both active and inactive KDs, as predicted by the idea that the asymmetric dimer mimics a trans‐autophosphorylation complex. These results support a model in which a structurally and functionally asymmetric, ‘front‐to‐front’ association mediates autophosphorylation of PknB and homologous kinases.     

以蛋白激酶B为靶点的新型抗结核药物高通量筛选模型的建立和应用

【目的】建立以结核分枝杆菌蛋白激酶B为靶点的高通量筛选模型,并运用此模型进行化合物的筛选。【方法】克隆和表达结核分枝杆菌蛋白激酶B,并以其为靶酶建立并优化PknB抑制剂高通量筛选模型,利用该模型对化合物样品进行筛选,并对筛选到的阳性化合物进行抗菌和抑酶活性评价。【结果】利用该模型筛选了化合物样品18 000个,得到具有抑酶活性的阳性化合物8个,其中3个化合物具有较好的对结核分枝杆菌、海分枝杆菌、耻垢分枝杆菌的抑菌活性。【结论】建立的以PknB为靶点的抗结核药物高通量筛选模型具有灵敏度高、稳定性强等优点,可成功用于化合物的高效筛选。筛选得到3个在抑酶水平和抗菌方面均具有良好活性的阳性化合物样品,值得进一步研究。     

Crystal structure of the catalytic domain of the PknB serine/threonine kinase from Mycobacterium tuberculosis

With the advent of the sequencing programs of prokaryotic genomes, many examples of the presence of serine/threonine protein kinases in these organisms have been identified. Moreover, these kinases could be classified as homologues of those belonging to the well characterized superfamily of the eukaryotic serine/threonine and tyrosine kinases. Eleven such kinases were recognized in the genome of Mycobacterium tuberculosis. Here we report the crystal structure of an active form of PknB, one of the four M. tuberculosis kinases that are conserved in the downsized genome of Mycobacterium leprae and are therefore presumed to play an important role in the processes that regulate the complex life cycle of mycobacteria. Our structure confirms again the extraordinary conservation of the protein kinase fold and constitutes a landmark that extends this conservation across the evolutionary distance between high eukaryotes and eubacteria. The structure of PknB, in complex with a nucleotide triphosphate analog, reveals an enzyme in the active state with an unprecedented arrangement of the Gly-rich loop associated with a new conformation of the nucleotide gamma-phosphoryl group. It presents as well a partially disordered activation loop, suggesting an induced fit mode of binding for the so far unknown substrates of this kinase or for some modulating factor(s).     

Substituted benzyl-pyrimidines targeting thymidine monophosphate kinase of Mycobacterium tuberculosis: synthesis and in vitro anti-mycobacterial activity

A series of N(1)-(4-substituted-benzyl)-pyrimidines were synthesized as potential inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). Key SAR parameters included the chain length substitution in para position of the benzyl ring, the functional group terminating the alkyl chain, and the substituent on the C-5 pyrimidine ring. Synthesized molecules were assayed against both recombinant enzyme and mycobacteria cultures. The most potent compounds have K(i) values in the micromolar range and an MIC(50) of 50microg/mL against Mycobacterium bovis. These results will guide the design of a new generation of lead compounds.     

Structure of Mycobacterium tuberculosis PknB supports a universal activation mechanism for Ser/Thr protein kinases

A family of eukaryotic-like Ser/Thr protein kinases occurs in bacteria, but little is known about the structures and functions of these proteins. Here we characterize PknB, a transmembrane signaling kinase from Mycobacterium tuberculosis. The intracellular PknB kinase domain is active autonomously, and the active enzyme is phosphorylated on residues homologous to regulatory phospho-acceptors in eukaryotic Ser/Thr kinases. The crystal structure of the PknB kinase domain in complex with an ATP analog reveals the active conformation. The predicted fold of the PknB extracellular domain matches the proposed targeting domain of penicillin-binding protein 2x. The structural and chemical similarities of PknB to metazoan homologs support a universal activation mechanism of Ser/Thr protein kinases in prokaryotes and eukaryotes.     

The Mycobacterium bovis homologous protein of the Mycobacterium tuberculosis serine/threonine protein kinase Mbk (PknD) is truncated

We previously identified a 70-kDa serine/threonine protein kinase (MbK or PknD) from Mycobacterium tuberculosis Erdman containing a transmembrane domain and bearing a 270-amino acid N-terminal kinase domain. With the use of a polyclonal serum, Mbk has now been identified by Western blotting in protein extracts from M. tuberculosis and confirmed to be localised in the envelope. An identical mbk gene has been found by sequencing different M. tuberculosis and M. africanum strains. Surprisingly, in two virulent M. bovis strains and four different strains of M. bovis BCG, an additional adenine after position 829 of the open reading frame was found that produces a frame shift resulting in a predicted truncated, presumably free cytoplasmic protein, encoding only the N-terminal 30-kDa Mbk kinase domain. This sequence polymorphism has been confirmed by Western blot analysis of M. bovis BCG protein extracts.     

Characterization of PDK2 activity against protein kinase B gamma

Protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase controlled by insulin, various growth factors, and phosphatidylinositol 3-kinase. Full activation of the PKB enzyme requires phosphorylation of a threonine in the activation loop and a serine in the C-terminal tail. PDK1 has clearly been shown to phosphorylate the threonine, but the mechanism leading to phosphorylation of the serine, the PDK2 site, is unclear. A yeast two-hybrid screen using full-length human PKBgamma identified protein kinase C (PKC) zeta, an atypical PKC, as an interactor with PKBgamma, an association requiring the pleckstrin homology domain of PKBgamma. Endogenous PKBgamma was shown to associate with endogenous PKCzeta both in cos-1 cells and in 3T3-L1 adipocytes, demonstrating a physiological interaction. Immunoprecipitates of PKCzeta, whether endogenous PKCzeta from insulin-stimulated 3T3-L1 adipocytes or overexpressed PKCzeta from cos-1 cells, phosphorylated S472 (the C-terminal serine phosphorylation site) of PKBgamma, in vitro. In vivo, overexpression of PKCzeta stimulated the phosphorylation of approximately 50% of the PKBgamma molecules, suggesting a physiologically meaningful effect. However, pure PKCzeta protein was incapable of phosphorylating S472 of PKBgamma. Antisense knockout studies and use of a PDK1 inhibitor showed that neither PKB autophosphorylation nor phosphorylation by PDK1 accounted for the S472 phosphorylation in PKCzeta immunoprecipitates. Staurosporine inhibited the PKCzeta activity but not the PDK2 activity in PKCzeta immunoprecipitates. Together these results indicate that an independent PDK2 activity exists that physically associates with PKCzeta and that PKCzeta, by binding PKBgamma, functions to deliver the PDK2 to a required location. PKCzeta thus functions as an adaptor, associating with a staurosporine-insensitive PDK2 enzyme that catalyzes the phosphorylation of S472 of PKBgamma. Because both PKCzeta and PKB have been proposed to be required for mediating a number of crucial insulin responses, formation of an active signaling complex containing PKCzeta, PKB, and PDK2 is an attractive mechanism for ensuring that all the critical sites on targets such as glycogen synthase kinase-3 are phosphorylated.     

9-Benzylpurines with inhibitory activity against Mycobacterium tuberculosis

9-Benzylpurines with a variety of substituents in the 2-, 6- and/or 8-position have been prepared and screened for antimycobacterial effects. High inhibitory activity against Mycobacterium tuberculosis was found for 9-benzylpurines carrying a phenylethynyl-, trans-styryl or aryl substituents in the 6-position and generally chlorine in the 2-position tends to increase activity.     

Peptidomimetic inhibitors of protein farnesyltransferase show potent antimalarial activity

Malaria continues to represent a very serious health problem in the tropics. The current methods of clinical treatment are showing deficiencies due to the increased incidence of resistance in the parasite. In the present paper we report the design, synthesis, and evaluation of potential antimalarial agents against a novel target, protein farnesyltransferase. We show that the most potent compounds are active against Plasmodium falciparum in vitro at submicromolar concentrations.     

Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1)

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.     

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study

The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation. Several pan-RSK inhibitors have been identified with BI-D1870 and the pseudo-analogs LJH685 and LJI308 being the most selective, potent, and frequently used small molecule inhibitors. We designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition. We have identified inhibitors of RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the MOLM-13 acute myeloid leukemia (AML) cell line. The results of our studies support that RSK2 inhibition can be achieved in MOLM-13 cells without potent cytotoxicity. The structure-activity data from this study will be used as a platform to develop novel RSK2 inhibitors.     

Structure-based prediction of Mycobacterium tuberculosis shikimate kinase inhibitors by high-throughput virtual screening

A structure-based virtual screening protocol was used to predict Mycobacterium tuberculosis shikimate kinase (MtSk) inhibitors. Docking simulations were performed using eHiTS software and 644 drug-like compounds were identified as potential inhibitors. Forty-two percent of such inhibitors had a structural relationship to a triazole or a tetrazole heteroaromatic system which may provide a candidate lead for the discovery of MtSk inhibitors.