Database searching for thymidine and thymidylate kinase inhibitors using three-dimensional structure-based methods

Structure-based drug design methods were used to search for novel inhibitors of herpes simplex virus type 1 (HSV-1) thymidine kinase and Mycobacterium tuberculosis thymidylate kinase. The method involved the use of crystal structure complexes to guide database searching for potential inhibitors. A number of weak inhibitors of HSV-2 were identified, one of which was found to inhibit HSV-1 TK and HSV-1 TK-deficient viral strains. Each compound tested against M. tuberculosis thymidylate kinase was found to have some activity. The best of these compounds was only 4.6-fold less potent than 3'-azido-3'-deoxythymidine-5'-monophosphate (AZTMP). This study demonstrates the utility of structure-based drug design methods in the search for novel enzyme inhibitors.     

Discovery of novel 3,5-disubstituted indole derivatives as potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases

A series of novel 3,5-disubstituted indole derivatives as potent and selective inhibitors of all three members of the Pim kinase family is described. High throughput screen identified a pan-Pim kinase inhibitor with a promiscuous scaffold. Guided by structure-based drug design, SAR of the series afforded a highly selective indole chemotype that was further developed into a potent set of compounds against Pim-1, 2, and 3 (Pim-1 and Pim-3: IC(50)≤2nM and Pim-2: IC(50)≤100nM).     

Characterization of pyrimidine deoxyribonucleoside kinase (thymidine kinase) and thymidylate kinase as a multifunctional enzyme in cells transformed by herpes simplex virus type 1 and in cells infected with mutant strains of herpes simplex virus.

Pyrimidine deoxyribonucleoside kinase (thymidine kinase [TK]) was purified from two herpes simplex virus type 1 (HVS-1)-transformed TK-deficient mouse (LMTK-) cell lines and from LMTK- cells infected with HSV-1 mutant viruses coding for variant TK enzymes. These preparations exhibited normal or variant virus-induced thymidylate kinase activities correlating with their relative TK activities. Neither virus-induced activity was detected in LMTK- cells infected with an HSV-1 TK-deficient mutant. These results suggest that HSV-1 thymidylate kinase activity and TK activity are mediated by the same protein.     

Aminoimidazo[1,2-a]pyridines as a new structural class of cyclin-dependent kinase inhibitors. Part 1: Design, synthesis, and biological evaluation

We have identified a novel structural class of protein serine/threonine kinase inhibitors comprised of an aminoimidazo[1,2-a]pyridine nucleus. Compounds from this family are shown to potently inhibit cyclin-dependent kinases by competing with ATP for binding to a catalytic subunit of the protein. Structure-based design approach was used to direct this chemical scaffold toward generating potent and selective CDK2 inhibitors. The discovery of this new class of ATP-site directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis of new medicinal chemistry tool in search for an effective treatment of cancer and other diseases that involve protein kinase signaling pathways.     

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.     

Synthesis and SAR of 2-phenoxypyridines as novel c-Jun N-terminal kinase inhibitors

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK3) inhibitors is described. The development and optimization of the 2-phenoxypyridine series was carried out from an earlier pyrimidine series of JNK1 inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.     

Study of Analogues of Thymidine-5′-Monophosphate and Thymidine as Substrates or Inhibitors of Chick Embryo Liver Thymidylate Kinase

Abstract

The phosphorylation of thymidine-5′-monophosphate (dTMP) by chick embryo liver thymidylate kinase (Km (dTMP) =1.2 μM) was inhibited by the 5′-monophosphate derivatives of 5-bromo-2′-deoxyuridine (5-Br-dUMP), 5-iodo-2′-deoxyuridine (5-I-dUMP), 2′,3′-dideoxythymidine (ddTMP), 3′-azido-3′-deoxythymidine (AZT-MP) and the methylene phosphonate analogue of AZT-MP with IC50 values of 8, 24, 14, 5 and 6 μM respectively. 5-Fluoro-2′-deoxyuridine (5-F-dUMP) and dUMP were poor inhibitors (IC50 values > 300 μM). 5-Br-dUMP and 5-I-dUMP were found to be significant substrates of thymidylate kinase with phosphorylation efficiencies (Vmax/Km) of 26 and 6% of that of dTMP, respectively. In contrast, AZT-MP and ddTMP were poor substrates, being phosphorylated 800-fold less efficiently than dTMP. Thymidylate kinase was also significantly inhibited by thymidine and AZT. Our data give a better insight into the topology of the dTMP binding site of this enzyme and show that the 3′-hydroxyl group of dTMP plays a critical role in catalysis.     

Herpes Simplex Virus Thymidine Kinase Gene-Transfected Tumor Cells; Sensitivity to Antiherpetic Drugs

Abstract

A series of antiherpetic 5-substituted 2′-deoxyuridine derivatives (i. e. BVDU) and guanine derivatives (i. e. ganciclovir) have been evaluated for their cytostatic activity against murine mammary carcinoma FM3A cell lines that are deficient in cytosol thymidine kinase, but transfected by the herpes simplex virus type 1 (HSV-1)- or type 2 (HSV-2)-specified thymidine kinase gene. Most compounds were endowed with a markedly higher cytostatic activity against the HSV TK gene-transfected tumor cells than against wild-type tumor cells. The principal target for cytostatic activity of the BVDU derivatives proved thymidylate synthase, whereas the guanine derivatives inhibited HSV TK gene-transfected tumor cell proliferation by competing with cellular DNA polymerase(s) and subsequent incorporation into the cellular genome.

     

Thymidine and thymidine-5'-O-monophosphate analogues as inhibitors of Mycobacterium tuberculosis thymidylate kinase

The affinity of a series of 2', 3'- and 5-modified thymidine analogues for Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) was evaluated. The affinities of several non-phosphorylated analogues are in the same order of magnitude as those of their phosphorylated congeners. In view of drug delivery problems associated with phosphorylated compounds, these 'free' nucleosides seem more promising leads in the search of TMPKmt inhibitors as novel anti-tuberculosis agents.     

The alphaherpesvirus US3/ORF66 protein kinases direct phosphorylation of the nuclear matrix protein matrin 3

The protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro, suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.     

Synthesis and SAR of 4-(pyrazol-3-yl)-pyridines as novel c-jun N-terminal kinase inhibitors

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK) inhibitors is described. The development of the 4-(pyrazol-3-yl)-pyridine series was discovered from an earlier pyrimidine series of JNK inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.     

Highly potent and selective 3-N-methylquinazoline-4(3H)-one based inhibitors of B-RafV600E kinase

Herein we describe the design of a novel series of ATP competitive B-Raf inhibitors via structure-based methods. These 3-N-methylquinazoline-4(3H)-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 16, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies. Our work also demonstrates that by replacing an aryl amide with an aryl sulfonamide, a multikinase inhibitor such as AZ-628, can be converted to a selective B-Raf inhibitor, a finding that should have broad application in kinase drug discovery.     

Discovery of novel tricyclic pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine derivatives as VEGFR-2 inhibitors

In an effort to develop ATP-competitive VEGFR-2 selective inhibitors, a novel series of tricyclic pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine derivatives were designed and synthesized. These compounds were characterized by IR, ¹H NMR, ¹³C NMR, elemental and mass spectral analyses. Docking studies have given a partial insight into the molecular determinants of the activity of this novel series in VEGFR-2 kinase active site. Moreover, these compounds were assessed at 10 μM for their selective inhibitory activities over a panel of 6 human kinases, namely VEGFR-1/Flt-1, VEGFR-2/KDR, EGFR, CDK5/p25, GSK3α and GSK3β. Compound N-(4,6-dimethylthieno[2,3-b]pyridine)-7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-amine (9d) exhibited the most potent and selective inhibitory activity against VEGFR-2/KDR over the six human kinases, with an IC₅₀ value 2.6 μM. The identification of this hit candidate could aid the design of new tricyclic-based VEGFR-2 kinase modulators.     

The A167Y mutation converts the herpes simplex virus type 1 thymidine kinase into a guanosine analogue kinase

The thymidine (dThd) kinase (TK) encoded by herpes simplex virus type 1 (HSV-1) is not only endowed with dThd kinase, but also with thymidylate (dTMP) kinase and 2'-deoxycytidine (dCyd) kinase (dCK) activity. HSV-1 TK also recognizes a variety of antiherpetic guanine nucleoside analogues such as acyclovir (ACV), ganciclovir (GCV), lobucavir (LBV), penciclovir (PCV), and others (i.e., A5021). Site-directed mutagenesis of the highly conserved Ala-167 to Tyr in HSV-1 TK completely abolished TK, dTMP-K, and dCK activity, but maintained ACV-, GCV-, LBV-, PCV-, and A5021-phosphorylating capacity. A variety of 5-substituted pyrimidine nucleoside substrates, but also a number of selective HSV-1 TK inhibitors structurally related to thymine lost significant binding affinity for the mutant enzyme and did not markedly compete with GCV phosphorylation by the mutant enzyme. These findings could be explained by computer-assisted modeling data that revealed steric hindrance of the pyrimidine ring in the HSV-1 TK active site by the large 4-hydroxybenzyl ring of 167-Tyr, while the positioning of the purine ring of guanine-based HIV-1 TK substrates in the active site was kept virtually unaltered. Surprisingly, the efficiency of conversion the antiherpetic 2'-deoxyguanosine analogues ACV, GCV, LBV, PCV, and A5021 to their phosphorylated forms by the A167Y mutant HSV-1 TK was far more pronounced than for the wild-type enzyme. Therefore, the single A167Y mutation converts the wild-type HSV-1 TK from a predominantly pyrimidine nucleos(t)ide kinase into a virtually exclusive purine (guanine) nucleoside analogue kinase.     

Biophysical and mechanistic insights into novel allosteric inhibitor of spleen tyrosine kinase

Extracellular stimulation of the B cell receptor or mast cell FcεRI receptor activates a cascade of protein kinases, ultimately leading to antigenic or inflammation immune responses, respectively. Syk is a soluble kinase responsible for transmission of the receptor activation signal from the membrane to cytosolic targets. Control of Syk function is, therefore, critical to the human antigenic and inflammation immune response, and an inhibitor of Syk could provide therapy for autoimmune or inflammation diseases. We report here a novel allosteric Syk inhibitor, X1, that is noncompetitive against ATP (K(i) 4 ± 1 μM) and substrate peptide (K(i) 5 ± 1 μM), and competitive against activation of Syk by its upstream regulatory kinase LynB (K(i) 4 ± 1 μM). The inhibition mechanism was interrogated using a combination of structural, biophysical, and kinetic methods, which suggest the compound inhibits Syk by reinforcing the natural regulatory interactions between the SH2 and kinase domains. This novel mode of inhibition provides a new opportunity to improve the selectivity profile of Syk inhibitors for the development of safer drug candidates.     

Carbocyclic thymidine derivatives efficiently inhibit Plasmodium falciparum thymidylate kinase (PfTMK)

During the course of our research into new anti-malaria drugs, Plasmodium falciparum thymidylate kinase (PfTMK) has emerged as an important drug target because of its unique substrate specificity. Compared with human thymidylate kinase (HsTMK), PfTMK shows broader substrate specificity, which includes both purine and pyrimidine nucleotides. PfTMK accepts both 2'-deoxyguanosine monophosphate (dGMP) and thymidine monosphosphate (TMP) as substrates. We have evaluated the inhibitory activity of seven carbocyclic thymidine analogs and report the first structure-activity relationship for these inhibitors against PfTMK. The 2',3' dideoxycarbocyclic derivative of thymidine showed the most potent inhibition of the enzyme. The K(i)(dTMP) and K(i)(dGMP) values were 20 and 7 μM respectively. Thus, further modifications of carbocyclic thymidine analogs represent a good strategy for developing more powerful thymidylate kinase inhibitors.     

An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases

The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.     

Potential application of thymidylate kinase in nucleoside analogue activation in Plasmodium falciparum

Plasmodium falciparum thymidylate kinase (PfTMPK) shows a broad range of substrate tolerance when compared to the corresponding human enzyme. Besides 2′-deoxythymidine monophosphate (dTMP), PfTMPK can phosphorylate 3′-azido-2′,3′-dideoxythymidine monophosphate (AZTMP) very efficiently. In contrast, human thymidylate kinase (hTMPK) is 200 times less active towards AZTMP. We were interested to see if we could use PfTMPK to activate 3′-azido-2′,3′-deoxythymidine (AZT) derivatives as a strategy to treat malaria. P. falciparum lacks a pyrimidine nucleoside kinase which usually activates nucleoside and nucleoside analogues to the corresponding monophosphates. Therefore, several prodrug analogues of AZT and related nucleoside monophosphates were prepared and analysed for antiparasitic activity. The prodrugs showed an increase in activity over the parent nucleoside analogues, which showed no inhibition of parasite growth at the concentration tested. The evidence here reported provides a strategy that could be exploited for further anti-malarial design.     

Conservative mutations of glutamine-125 in herpes simplex virus type 1 thymidine kinase result in a ganciclovir kinase with minimal deoxypyrimidine kinase activities

The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is the major anti-herpes virus pharmacological target, and it is being utilized in combination with the prodrug ganciclovir as a toxin gene therapeutic for cancer. One active-site amino acid, glutamine-125 (Gln-125), has been shown to form hydrogen bonds with bound thymidine, thymidylate, and ganciclovir in multiple X-ray crystal structures. To examine the role of Gln-125 in HSV-1 TK activity, three site-specific mutations of this residue to an aspartic acid, an asparagine, or a glutamic acid were introduced. These three mutants and wild-type HSV-1 TK were expressed in E. coli and partially purified and their enzymatic properties compared. In comparison to the Gln-125 HSV-1 TK, thymidylate kinase activity of all three mutants was decreased by over 90%. For thymidine kinase activity relative to Gln-125 enzyme, the K(m) of thymidine increased from 0.9 microM for the parent Gln-125 enzyme to 3 microM for the Glu-125 mutant, to 6000 microM for the Asp-125 mutant, and to 20 microM for the Asn-125 mutant. In contrast, the K(m) of ganciclovir decreased from 69 microM for the parent Gln-125 enzyme to 50 microM for the Asn-125 mutant and increased to 473 microM for the Glu-125 mutant. The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, but with nonlinear kinetics. Molecular simulations of the wild-type and mutant HSV-1 TK active sites predict that the observed activities are due to loss of hydrogen bonding between thymidine and the mutant amino acids, while the potential for hydrogen bonding remains intact for ganciclovir binding. When expressed in two mammalian cell lines, the Glu-125 mutant led to GCV-mediated killing of one cell line, while the Asn-125 mutant was equally as effective as wild-type HSV-1 TK in metabolizing GCV and causing cell death in both cell lines.     

Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding

Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylate kinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix alpha3 shifting position initially, followed by movement of alpha2 to close the substrate site. In addition, we observe significant conformational differences in the TMP-binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to the thymine ring, whereas a cis-proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species.