Using fragment cocktail crystallography to assist inhibitor design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase

The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SADa phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited "fragment cocktail soaks". Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of approximately 10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins.     

Phosphorylation of 9-(2-phosphonomethoxyethyl)adenine and 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine by AMP(dAMP) kinase from L1210 cells.

Acyclic nucleotide analogues 9-(2-phosphonomethoxyethyl)adenine (PMEA) and 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine ((S)-HPMPA) which display potent antiviral activity are transformed in the cells to their mono- and disphosphoryl derivatives. We purified from mouse L1210 cells the enzyme that in two steps phosphorylates PMEA and (S)-HPMPA to their diphosphoryl derivatives and found that it co-purifies with AMP(dAMP) kinase activity; the best substrates of this enzyme were AMP, ADP and dAMP. Other nucleoside 5'-triphosphates or creatine phosphate could not be substituted for ATP as a phosphate donor. Our results also indicated that at least one other enzyme (creatine kinase) is capable of transforming the monophosphoryl derivatives of the studied compounds to their respective diphosphates.     

Enzyme fragment complementation binding assay for p38alpha mitogen-activated protein kinase to study the binding kinetics of enzyme inhibitors

The majority of protein kinase assays used in drug discovery research are enzyme activity assays. These assays are based on the measurement of phosphorylated protein or peptide substrate, which is the end product of the enzyme reaction. Since most kinase inhibitors are ATP competitive, prediction of the activity of compounds in cellular systems based on potency values in enzyme activity assays is complex, as this should take into account the affinity of the enzyme for ATP and the cellular ATP concentration. The fact that some of the most successful kinase inhibitors, such as STI 571 (imatinib mesylate, Gleevec, Novartis Pharmaceuticals, East Hanover, NJ), act through binding to the inactive isoform of the kinase provides another limitation of enzyme activity assays. Binding assays allow separate measurement of compound affinity to active and inactive kinase and do not require ATP or substrate in the reaction. Recently, a non-radioactive kinase binding assay for p38 mitogen-activated protein kinase has become available from DiscoveRx (Fremont, CA). The assay method, called HitHunter, utilizes enzyme fragment complementation of Escherichia coli beta-galactosidase to generate an assay signal by chemiluminescence. We have reconfigured the commercial assay kit to study the binding kinetics of two known reference inhibitors of the alpha-isoform of p38, the pyridinyl imidazole SB 203580 and the diaryl urea BIRB 796. Our data confirm the slow association kinetics of BIRB 796 as compared to SB 203580, which corresponded with the requirement of a relatively long preincubation time to obtain maximal effect in a cellular assay. Although neither of the two compounds showed preference for either active or inactive p38alpha, our data demonstrate that the HitHunter kinase binding assay can be used to select compounds that specifically target inactive kinase.     

Analysis of c‐Met Kinase Domain Complexes: A New Specific Catalytic Site Receptor Model for Defining Binding Modes of ATP‐Competitive Ligands

The receptor tyrosine kinase c‐Met have multiple roles during cancer development and is currently considered as an important target for molecularly targeted therapies. Structural knowledge of how compounds interact on c‐Met catalytic site could guide structure‐based drug design strategies towards more effective and selective anticancer drug candidates. However, although 17 crystal structures of c‐Met complexed with adenosine triphosphate (ATP)‐competitive kinase inhibitors are publicly available (August 2009), there are still open questions regarding the prediction of ligand binding modes. We have applied molecular modeling and molecular mechanics to analyze the distribution of ligands interaction energy on c‐Met residues, and deduced a new model of the active site allowing for an unambiguous identification of ligand binding modes. We demonstrate that the binding of known ligands on the c‐Met catalytic site involves seven identified structurally‐distinct areas. Five of these match the generic kinase ATP binding site model built by Novartis scientists in the 1990s, while the two others are distinct allosteric regions that can be exploited by second generation kinase inhibitors such as Gleevec. We show here that c‐Met can accept both such kinds of allosteric inhibitors, a very unusual feature in the kinase family that opens new grounds for highly specific drug design.     

Salicylate inhibition of monkey muscle creatine kinase

Creatine kinase is inhibited in both the forward and back reactions by salicylate compounds. The inhibition process is complex, resulting in curved Dixon plots, and is non-competitive with respect to creatine. Salicylate does not affect the inhibition caused by chloride ions but reduces the activation by acetate. The ionizations responsible for the pH/activity curve are also suppressed. These results are interpreted to indicate that more than one salicylate molecule binds per active centre at a site away from that for the transferable phosphoryl group. Inhibition probably involves binding to a particular enzyme conformational state that also prevents activation by acetate.     

Dissociation,unfolding and inactivation of creatine kinase in urea solutions

It was previously reported that, during unfolding of creatine kinase in guanidinium chloride or urea solutions, inactivation occured before noticeable conformational change could be detected, suggesting that the conformation at the active site is more easily perturbed and, hence, more flexible than the molecule as a whole [Tsou (1986) Trends Biochem. Sci. 11 , 417–429]. In the present paper, the urea-gradient electrophoresis and the isoenzyme hybrid of creatine kinase has been studied. The results show that at low urea concentrations, creatine kinase is still in the dimeric state or only slightly dissociated. The dissociation and inactivation of creatine kinase during denaturation by urea are also compared. It was found that the enzyme was nearly inactivated in low urea concentrations before noticeable dissociation was detected. It therefore appears that in low urea concentrations, inactivation of creatine kinase is not due to the dissociation of the active dimer. The present result supports the hypothesis of the conformational flexibility of the active site in this enzyme. © Munksgaard 1996.     

Kinetic studies on the inhibition of creatine kinase activity by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one in the cerebral cortex of rats

Tellurium has been used as an industrial component of many alloys and in the electronic industry. Organotellurium compounds can cause poisoning which leads to neurotoxic symptoms such as significant impairment of learning, spatial memory and are potentially neurotoxic to human beings. However, the molecular mechanisms of neurotoxicity of organotellurium compounds are not well understood. Considering that creatine kinase plays a key role in energy metabolism of tissues with intermittently high and fluctuating energy requirements, such as nervous tissue, the main objective of this study was to investigate the mechanisms by which 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one inhibit creatine kinase activity, a key enzyme of energy homeostasis, in the cerebral cortex of 30-day-old Wistar rats. For the kinetic studies, the Lineweaver–Burk plot was used to characterize the mechanisms of enzyme inhibition by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one. The results suggested that this compound inhibits creatine kinase activity by two different mechanisms: competition with ADP and oxidation of critical sulfhydryl groups for the functioning of the enzyme. The potential for inhibition of creatine kinase to occur in vivo may contribute to the neurotoxicity observed by this organochaocogen.     

Multiple binding sites for substrates and modulators of semicarbazide-sensitive amine oxidases: kinetic consequences

Human semicarbazide-sensitive amine oxidase (SSAO) is a target for novel anti-inflammatory drugs that inhibit enzymatic activity. However, progress in developing such drugs has been hampered by an incomplete understanding of mechanisms involved in substrate turnover. We report here results of a comparative study of human and bovine SSAO enzymes that reveal binding of substrates and other ligands to at least two (human) and up to four (bovine) distinct sites on enzyme monomers. Anaerobic spectroscopy reveals binding of substrates (spermidine and benzylamine) and of an imidazoline site ligand (clonidine) to the reduced active site of bovine SSAO, whereas interactions with oxidized enzyme are evident in kinetic assays and crystallization studies. Radioligand binding experiments with [(3)H]tetraphenylphosphonium, an inhibitor of bovine SSAO that binds to an anionic cavity outside the active site, reveal competition with spermidine, benzylamine, and clonidine, indicating that these ligands also bind to this second anionic region. Kinetic models of bovine SSAO are consistent with one spermidine molecule straddling the active and secondary sites on both oxidized and reduced enzyme, whereas these sites are occupied by two individual molecules of smaller substrates such as benzylamine. Clonidine and other imidazoline site ligands enhance or inhibit activity as a result of differing affinities for both sites on oxidized and reduced enzyme. In contrast, although analyses of kinetic data obtained with human SSAO are also consistent with ligands binding to oxidized and reduced enzyme, we observed no apparent requirement for substrate or modulator binding to any secondary site to model enzyme behavior.     

Application of fragment screening and fragment linking to the discovery of novel thrombin inhibitors

The screening of fragments is an alternative approach to high-throughput screening for the identification of leads for therapeutic targets. Fragment hits have been discovered using X-ray crystallographic screening of protein crystals of the serine protease enzyme thrombin. The fragment library was designed to avoid any well-precedented, strongly basic functionality. Screening hits included a novel ligand (3), which binds exclusively to the S2-S4 pocket, in addition to smaller fragments which bind to the S1 pocket. The structure of these protein-ligand complexes are presented. A chemistry strategy to link two such fragments together and to synthesize larger drug-sized compounds resulted in the efficient identification of hybrid inhibitors with nanomolar potency (e.g., 7, IC50 = 3.7 nM). These potent ligands occupy the same area of the active site as previously described peptidic inhibitors, while having very different chemical architecture.     

Factors influencing the specificity of inhibitor binding to the human and malaria parasite dihydroorotate dehydrogenases

The de novo pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase is an emerging drug target for the treatment of malaria. In this context a key property of Plasmodium falciparum DHODH (PfDHODH) is that it can be selectively inhibited over its human homologue (HsDHODH). However, HsDHODH is also a validated drug target for autoimmune diseases such as arthritis. Here a series of novel inhibitors is described that includes compounds that switch specificity between the two enzymes as a result of small alterations in chemical structure. Structure-activity relationship (SAR), crystallography, docking, and mutagenesis studies are used to examine the binding modes of the compounds within the two enzymes and to reveal structural changes induced by inhibitor binding. Within this series, compounds with therapeutically relevant HsDHODH activity are described and their binding modes characterized using X-ray crystallography, which reveals a novel conformational shift within the inhibitor binding site.     

Identification of novel small molecule ligands that bind to tetanus toxin

Tetanus toxin belongs to a family of clostridial protein neurotoxins for which there are no known antidotes. Another closely related member of this family, botulinum toxin, is being used with increasing frequency by physicians to treat severe muscle disorders. Botulinum toxin has also been produced in large quantities by terrorists for use as a biological weapon. To identify small molecule ligands that might bind to the targeting domain of tetanus and botulinum toxins and to facilitate the design of inhibitors and new reagents for their detection, molecular docking calculations were used to screen a large database of compounds for their potential to bind to the C fragment of tetanus toxin. Eleven of the predicted ligands were assayed by electrospray ionization mass spectrometry (ESI-MS) for binding to the tetanus toxin C fragment, and five ligands (45%) were found to bind to the protein. One of these compounds, doxorubicin, was observed to have strong hydrophobic interactions with the C fragment. To check the ligands for their ability to compete with ganglioside binding, each was also tested using a GT1b liposome assay. Doxorubicin was the only ligand found to competitively bind the tetanus toxin C fragment with an appreciable binding constant (9.4 microM).     

Protein-ligand crystal structures can guide the design of selective inhibitors of the FGFR tyrosine kinase

The design of compounds that selectively inhibit a single kinase is a significant challenge, particularly for compounds that bind to the ATP site. We describe here how protein-ligand crystal structure information was able both to rationalize observed selectivity and to guide the design of more selective compounds. Inhibition data from enzyme and cellular screens and the crystal structures of a range of ligands tested during the process of identifying selective inhibitors of FGFR provide a step-by-step illustration of the process. Steric effects were exploited by increasing the size of ligands in specific regions in such a way as to be tolerated in the primary target and not in other related kinases. Kinases are an excellent target class to exploit such approaches because of the conserved fold and small side chain mobility of the active form.     

Design and synthesis of photoaffinity-labeling ligands of the L-prolyl-L-leucylglycinamide binding site involved in the allosteric modulation of the dopamine receptor

Pro-Leu-Gly-NH(2) (PLG), in addition to its endocrine effects, possesses the ability to modulate dopamine D(2) receptors within the central nervous system. However, the precise binding site of PLG is unknown. Potential photoaffinity-labeling ligands of the PLG binding site were designed as tools to be used in the identification of the macromolecule that possesses this binding site. Six different photoaffinity-labeling ligands were designed and synthesized on the basis of the gamma-lactam PLG peptidomimetic 1. The 4-azidobenzoyl and 4-azido-2-hydroxybenzoyl photoaffinity-labeling moieties were placed at opposite ends of PLG peptidomimetic 1 to generate a series of ligands that potentially could be used to map the PLG binding site. All of the compounds that were synthesized possessed activity comparable to or better than PLG in enhancing [(3)H]-N-propylnorapomorphine agonist binding to dopamine receptors. Photoaffinity ligands that were cross-linked to the receptor preparation produced a modulatory effect that was either comparable to or greater than the increase in agonist binding produced by the respective ligands that were not cross-linked to the dopamine receptor. The results indicate that these photoaffinity-labeling agents are binding at the same allosteric site as PLG and PLG peptidomimetic 1.     

Kinetic analysis of protein kinase C inhibition by staurosporine: evidence that inhibition entails inhibitor binding at a conserved region of the catalytic domain but not competition with substrates.

The indole carbazole staurosporine is an extraordinarily potent antiproliferative agent that inhibits the growth of cultured mammalian cells at concentrations of less than 1 nM. The antiproliferative activity of staurosporine is attributed to its potent inhibition of diverse protein kinases, but the mechanism of staurosporine inhibition has not been elucidated for any protein kinase. Protein kinase C (PKC) is a family of Ca(2+)- and phosphatidylserine-dependent protein kinases that are activated in vivo by the second messenger diacylglycerol. A fully active, Ca(2+)- and phosphatidylserine-independent, catalytic fragment of PKC that contains only the catalytic domain of the enzyme can be produced by limited proteolysis. Previous studies indicated that staurosporine inhibits PKC by binding its catalytic domain. In this study, we define the kinetics of inhibition by staurosporine of a catalytic fragment of rat brain PKC-gamma and of a catalytic fragment generated from a rat brain PKC-alpha/PKC-beta mixture. Our kinetic results provide evidence that staurosporine inhibits PKC by binding to a site of the catalytic domain other than the ATP substrate and protein substrate binding sites. Staurosporine inhibition appears to entail binding at a conserved site in the catalytic domain of PKC, because staurosporine inhibited rat brain PKC-alpha, PKC-beta, and PKC-gamma, as well as the catalytic fragments of PKC-beta and PKC-gamma, with similar protencies. The kinetics of inhibition of the catalytic fragment of PKC-gamma were uncompetitive with respect to histone III-S, providing evidence that the binding of histone III-S at the active site of the catalytic fragment precedes the binding of staurosporine to the enzyme. Taken in the context of previous mechanistic studies of PKC-catalyzed histone III-S phosphorylation, these results provide evidence that staurosporine binds to a complex of PKC, MgATP, and histone III-S, thereby forming a complex that cannot break down to products. In addition, the inhibitory kinetics observed when the ATP concentration was varied provided evidence that staurosporine reduces the affinity of MgATP for the catalytic fragment of PKC-gamma. Thus, the kinetics of inhibition of the catalytic fragment of PKC-gamma by staurosporine provide evidence that staurosporine inhibits PKC by a mixed mechanism.     

Cysteine mapping in conformationally distinct kinase nucleotide binding sites: application to the design of selective covalent inhibitors

Kinases have emerged as one of the most prolific therapeutic targets. An important criterion in the therapeutic success of inhibitors targeting the nucleotide binding pocket of kinases is the inhibitor residence time. Recently, covalent kinase inhibitors have attracted attention since they confer terminal inhibition and should thus be more effective than reversible inhibitors with transient inhibition. The most robust approach to design irreversible inhibitors is to capitalize on the nucleophilicity of a cysteine thiol group present in the target protein. Herein, we report a systematic analysis of cysteine residues present in the nucleotide binding site of kinases, which could be harnessed for irreversible inhibition, taking into consideration the different kinase conformations. We demonstrate the predictive power of this analysis with the design and validation of an irreversible inhibitor of KIT/PDGFR kinases. This is the first example of a covalent kinase inhibitor that combines a pharmacophore addressing the DFG-out conformation with a covalent trap.     

Structure-activity relationship for adenosine kinase from Mycobacterium tuberculosis II. Modifications to the ribofuranosyl moiety

Adenosine kinase (Ado kinase) from Mycobacterium tuberculosis is structurally and biochemically unique from other known Ado kinases. This purine salvage enzyme catalyzes the first step in the conversion of the adenosine analog, 2-methyl-Ado (methyl-Ado), into a metabolite with antitubercular activity. Methyl-Ado has provided proof of concept that the purine salvage pathway from M. tuberculosis may be utilized for the development of antitubercular compounds with novel mechanisms of action. In order to utilize this enzyme, it is necessary to understand the topography of the active site to rationally design compounds that are more potent and selective substrates for Ado kinase. A previous structure-activity relationship identified modifications to the base moiety of adenosine (Ado) that result in substrate and inhibitor activity. In an extension of that work, 62 Ado analogs with modifications to the ribofuranosyl moiety, modifications to the base and ribofuranosyl moiety, or modifications to the glycosidic bond position have been analyzed as substrates and inhibitors of M. tuberculosis Ado kinase. A subset of these compounds was further analyzed in human Ado kinase for the sake of comparison. Although no modifications to the ribose moiety resulted in compounds as active as Ado, the best substrates identified were carbocyclic-Ado, 8-aza-carbocyclic-Ado, and 9-[alpha-l-lyxofuranosyl]-adenine with 38%, 4.3%, and 3.8% of the activity of Ado, respectively. The most potent inhibitor identified, 5'-amino-5'-deoxy-Ado, had a K(i)=0.8muM and a competitive mode of inhibition. MIC studies demonstrated that poor substrates could still have potent antitubercular activity.     

Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action

The effects of inhibitors of protein kinase C on the activities of the intact enzyme, the proteolytically-generated catalytic domain, and [3H]phorbol 12,13-dibutyrate (PDBu) binding were compared in an effort to evaluate this approach for clarifying mechanisms of inhibitor action. Staurosporine, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], and quercetin inhibited the catalytic fragment with similar potencies as for the intact enzyme while having little or no effect on binding, consistent with reports that they are competitive with ATP. Adriamycin, trifluoperazine, and tamoxifen, suggested to disrupt hydrophobic interactions between the regulatory domain of protein kinase C and phospholipid, were all most effective on the intact enzyme. They appear to possess a mixed mechanism, however, inhibiting activity of the catalytic domain with approximately 3-fold lower potencies. Gossypol inhibited intact enzyme, catalytic fragment, and PDBu binding with similar potencies. In light of multiple apparent sites of action for such protein kinase C inhibitors, comparison of their activities on the individual functional domains of the kinase may provide a useful complement to studies with the intact enzyme.     

Kinase-likeness and kinase-privileged fragments: toward virtual polypharmacology

Small molecule protein kinase inhibitors are widely employed as biological reagents and as leads in the design of drugs for a variety of diseases. We investigated the phenomenon of kinase-likeness, i.e., the propensity of ligands to inhibit protein kinases, in the context of kinase-specific substructural fragments. The frequency of occurrence of multiple structural fragments in kinase inhibitor libraries relative to nonkinase compounds has been analyzed. A combination of structural fragment counts, termed the "2-0" kinase-likeness rule, provides approximately 5-fold enrichment in kinase active compounds. This rule has been validated using in-house kinase counterscreening data and applied prospectively to uncover kinase activities in marketed drugs. In addition, the role of discriminating fragments in kinase recognition was interrogated using available structural data, providing an insight into their effect on inhibitor potency and selectivity. One of these fragments, bisarylaniline, has been characterized as a kinase-privileged fragment with specific binding preferences and a link to increased activity within kinases.     

Characterization of the melatoninergic MT3 binding site on the NRH:quinone oxidoreductase 2 enzyme

Melatonin acts through a series of molecular targets: the G-protein coupled receptors, MT1 and MT2, and a third binding site, MT3, recently identified as the enzyme NRH:quinone oxydoreductase 2 (QR2). The relationship between the multiple physiological functions of melatonin and this enzyme remains unclear. Because of the relationship of QR2 with the redox status of cells, these studies could bring the first tools for a molecular rationale of the antioxidant effects of melatonin. In the present paper, we used a QR2-stably expressing cell line and hamster kidneys to compare the 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine binding data, and to characterize the MT3 binding site. We designed and tested compounds from two distinct chemicals series in a displacement assay of the two MT3 ligands, 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine from their cloned target. We also tested their ability to inhibit QR2 catalytic activity. These compounds were separated into two classes: those that bind within the catalytic site (and being inhibitors) and those that bind outside it (and therefore not being inhibitors). Compounds range from potent ligands (K(i) = 1 nM) to potent inhibitors (14 nM), and include one compound [NMDPEF: N-[2-(2-methoxy-6H-dipyrido[2,3-a:3,2-e]pyrrolizin-11-yl)ethyl]-2-furamide] active on both parameters in the low nanomolar range. To dissect the physio-pathological pathways in which QR2, MT3 and melatonin meet, one needs more compounds binding to MT3 and/or inhibitors of QR2 enzymatic activity. The compounds described in the present paper are new tools for such a task.     

A Back-to-Front Fragment-Based Drug Design Search Strategy Targeting the DFG-Out Pocket of Protein Tyrosine Kinases

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