Exploring acyclic nucleoside analogues as inhibitors of Mycobacterium tuberculosis thymidylate kinase

In the search for novel inhibitors of the enzyme thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt), an attractive target for novel antituberculosis agents, we report herein the discovery of the first acyclic nucleoside analogues that potently and selectively inhibit TMPKmt. The most potent compounds in this series are (Z)-butenylthymines carrying a naphtholactam or naphthosultam moiety at position 4, which display K(i) values of 0.42 and 0.27 microM, respectively. Docking studies followed by molecular dynamics simulations performed to rationalize the interaction of this new family of inhibitors with the target enzyme revealed a key interaction between the distal substituent and Arg 95 in the target enzyme. The fact that these inhibitors are more easily synthesizable than previously identified TMPKmt inhibitors, together with their potency against the target enzyme, makes them attractive lead compounds for further optimization.     

Metabolic labeling of DNA by purine analogues in vivo

DNA tracking: The 2'-deoxyadenosine analogue "EdA" efficiently labels newly synthesized DNA with alkyne groups in vivo without inhibiting cell cycle progression or embryonic development. This provides the first demonstration that a bio-orthogonal functional group can be metabolically incorporated into DNA through endogenous pathways by addition of a purine analogue to cells.     

微波与超声波提取贻贝中嘌呤物质的方法研究

以贻贝为原料,用微波和超声波对贻贝中嘌呤提取工艺进行了研究,探讨了两种方法的最佳提取条件和参数。结果表明,微波辅助萃取最佳工艺参数为:液料质量比40∶1,萃取时间为20 min,萃取温度65℃,嘌呤总提取率达1.695 8 mg/g;超声波提取条件为液料质量比80∶1,提取150 min,嘌呤总提取率为1.681 6 mg/g。与超声波提取方法相比,微波辅助萃取法具有萃取时间短、提取率高、节省溶剂等优点。     

Design, synthesis, and evaluation of 5'-diphenyl nucleoside analogues as inhibitors of the Plasmodium falciparum dUTPase

Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a potential drug target for malaria. We previously reported some 5'-tritylated deoxyuridine analogues (both cyclic and acyclic) as selective inhibitors of the Plasmodium falciparum dUTPase. Modelling studies indicated that it might be possible to replace the trityl group with a diphenyl moiety, as two of the phenyl groups are buried, whereas the third is exposed to solvent. Herein we report the synthesis and evaluation of some diphenyl analogues that have lower lipophilicity and molecular weight than the trityl lead compound. Co-crystal structures show that the diphenyl inhibitors bind in a similar manner to the corresponding trityl derivatives, with the two phenyl moieties occupying the predicted buried phenyl binding sites. The diphenyl compounds prepared show similar or slightly lower inhibition of PfdUTPase, and similar or weaker inhibition of parasite growth than the trityl compounds.     

Synthesis and biological evaluation of bicyclic nucleosides as inhibitors of M. tuberculosis thymidylate kinase

Herein we describe the synthesis and conformational analysis of a series of bicyclic thymidine derivatives and their evaluation as inhibitors of thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), based on previously discovered bicyclic sugar nucleosides. With a K(i) value of 2.3 microm, 1-[3-aminomethyl-3,5-dideoxy-2-O,6-N-(thiocarbonyl)-beta-D-ribofuranosyl]thymine emerged as the most potent TMPK inhibitor of this series. Moreover, this promising compound displays inhibitory potency against Mycobacteria cultures with an IC(99) value of 100 microg mL(-1), thus promoting TMPKmt for the first time as a validated target for further inhibitory design. Attempts to rationalise the observed structure-activity relationship (SAR) involving molecular modelling and conformational analysis are described.     

Influence of sugar ring conformation on the transportability of nucleosides by human nucleoside transporters

The conformational preference of human nucleoside transporters (hNTs) with respect to sugar ring was examined using conformationally fixed purine and pyrimidine nucleosides built on a bicyclo[3.1.0]hexane template. These fixed-conformation nucleosides, methanocarba-deoxyadenosine or methanocarba-deoxycytidine in North (C3'-endo, N-MCdA and N-MCdC) or South (C2'-endo, S-MCdA and S-MCdC) conformations, were used to study inhibition of equilibrative (hENT1-4) and concentrative (hCNT1-3) nucleoside transport by individual recombinant hNTs produced in Saccharomyces cerevisiae cells or Xenopus laevis oocytes. Our results indicated that nucleosides in the North conformation were potent inhibitors of transport mediated by hCNTs whereas South nucleosides were inhibitors of hENTs, thus showing differences in the interaction with the hNTs. In summary, hCNTs exhibited strong preferences for North nucleosides whereas hENTs exhibited slight preferences for South nucleosides, demonstrating for the first time different conformational preferences among members of the two families of hNTs.     

Nanomolar competitive inhibitors of Mycobacterium tuberculosis and Streptomyces coelicolor type II dehydroquinase

Isomeric nitrophenyl and heterocyclic analogues of the known inhibitor (1S,3R,4R)-1,3,4-trihydroxy-5-cyclohexene-1-carboxylic acid have been synthesized and tested as inhibitors of M. tuberculosis and S. coelicolor type II dehydroquinase, the third enzyme of the shikimic acid pathway. The target compounds were synthesized by a combination of Suzuki and Sonogashira cross-coupling and copper(I)-catalyzed 2,3-dipolar cycloaddition reactions from a common vinyl triflate intermediate. These studies showed that a para-nitrophenyl derivative is almost 20-fold more potent as a competitive inhibitor against the S. coelicolor enzyme than that of M. tuberculosis. The opposite results were obtained with the meta isomer. Five of the bicyclic analogues reported herein proved to be potent competitive inhibitors of S. coelicolor dehydroquinase, with inhibition constants in the low nanomolar range (4-30 nM). These derivatives are also competitive inhibitors of the M. tuberculosis enzyme, but with lower affinities. The most potent inhibitor against the S. coelicolor enzyme, a 6-benzothiophenyl derivative, has a K(i) value of 4 nM-over 2000-fold more potent than the best previously known inhibitor, (1R,4R,5R)-1,5-dihydroxy-4-(2-nitrophenyl)cyclohex-2-en-1-carboxylic acid (8 microM), making it the most potent known inhibitor against any dehydroquinase. The binding modes of the analogues in the active site of the S. coelicolor enzyme (GOLD 3.0.1), suggest a key pi-stacking interaction between the aromatic rings and Tyr 28, a residue that has been identified as essential for enzyme activity.     

Interaction of the catalytic domain of inositol 1,4,5-trisphosphate 3-kinase A with inositol phosphate analogues

The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D-2-deoxy-Ins(1,4,5)P3 was devised. D-2-Deoxy-Ins(1,4,5)P3 and D-3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D-2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.     

海嘧啶对小白鼠S_(180)、EAC和H_(22)肿瘤细胞膜钠泵活性的影响

目的观察海嘧啶对小白鼠实体型肿瘤(S180)和腹水型肿瘤(EAC,H22)细胞膜钠泵活性的影响。方法荷瘤小鼠分为治疗组和对照组,分别测定各组瘤细胞膜钠泵活性。结果海嘧啶对小鼠S180、EAC及H22肿瘤细胞膜钠泵活性有明显的抑制作用。结论海嘧啶抗肿瘤的药理机制与其能够抑制肿瘤细胞膜钠泵活性有关。     

Inhibition of Mycobacterium tuberculosis Transaminase BioA by Aryl Hydrazines and Hydrazides

7,8‐Diaminopelargonic acid synthase (BioA) of Mycobacterium tuberculosis is a recently validated target for therapeutic intervention in the treatment of tuberculosis (TB). Using biophysical fragment screening and structural characterization of compounds, we have identified a potent aryl hydrazine inhibitor of BioA that reversibly modifies the pyridoxal‐5′‐phosphate (PLP) cofactor, forming a stable quinonoid. Analogous hydrazides also form covalent adducts that can be observed crystallographically but are incapable of inactivating the enzyme. In the X‐ray crystal structures, small molecules induce unexpected conformational remodeling in the substrate binding site. We compared these conformational changes to those induced upon binding of the substrate (7‐keto‐8‐aminopelargonic acid), and characterized the inhibition kinetics and the X‐ray crystal structures of BioA with the hydrazine compound and analogues to unveil the mechanism of this reversible covalent modification.     

Design and Structural Analysis of Aromatic Inhibitors of Type II Dehydroquinase from Mycobacterium tuberculosis

3‐Dehydroquinase, the third enzyme in the shikimate pathway, is a potential target for drugs against tuberculosis. Whilst a number of potent inhibitors of the Mycobacterium tuberculosis enzyme based on a 3‐dehydroquinate core have been identified, they generally show little or no in vivo activity, and were synthetically complex to prepare. This report describes studies to develop tractable and drug‐like aromatic analogues of the most potent inhibitors. A range of carbon–carbon linked biaryl analogues were prepared to investigate the effect of hydrogen bond acceptor and donor patterns on inhibition. These exhibited inhibitory activity in the high‐micromolar range. The addition of flexible linkers in the compounds led to the identification of more potent 3‐nitrobenzylgallate‐ and 5‐aminoisophthalate‐based analogues.     

Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages

The enzyme Zmp1 is a zinc‐containing peptidase that plays a critical role in the pathogenicity of Mycobacterium tuberculosis. Herein we describe the identification of a small set of Zmp1 inhibitors based on a novel 8‐hydroxyquinoline‐2‐hydroxamate scaffold. Among the synthesized compounds, N‐(benzyloxy)‐8‐hydroxyquinoline‐2‐carboxamide ( 1 c ) was found to be the most potent Zmp1 inhibitor known to date, and its binding mode was analyzed both by kinetics studies and molecular modeling, identifying critical interactions of 1 c with the zinc ion and residues in the active site. The effect of 1 c on intracellular Mycobacterium survival was assayed in J774 murine macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and human monocyte‐derived macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and genotoxicity were also assessed. Overall, inhibitor 1 c displays interesting in vitro antitubercular properties worthy of further investigation.     

Repurposing the Chemical Scaffold of the Anti‐Arthritic Drug Lobenzarit to Target Tryptophan Biosynthesis in Mycobacterium tuberculosis

The emergence of extensively drug‐resistant strains of Mycobacterium tuberculosis (Mtb) highlights the need for new therapeutics to treat tuberculosis. We are attempting to fast‐track a targeted approach to drug design by generating analogues of a validated hit from molecular library screening that shares its chemical scaffold with a current therapeutic, the anti‐arthritic drug Lobenzarit (LBZ). Our target, anthranilate phosphoribosyltransferase (AnPRT), is an enzyme from the tryptophan biosynthetic pathway in Mtb. A bifurcated hydrogen bond was found to be a key feature of the LBZ‐like chemical scaffold and critical for enzyme inhibition. We have determined crystal structures of compounds in complex with the enzyme that indicate that the bifurcated hydrogen bond assists in orientating compounds in the correct conformation to interact with key residues in the substrate‐binding tunnel of Mtb‐AnPRT. Characterising the inhibitory potency of the hit and its analogues in different ways proved useful, due to the multiple substrates and substrate binding sites of this enzyme. Binding in a site other than the catalytic site was found to be associated with partial inhibition. An analogue, 2‐(2‐5‐methylcarboxyphenylamino)‐3‐methylbenzoic acid, that bound at the catalytic site and caused complete, rather than partial, inhibition of enzyme activity was found. Therefore, we designed and synthesised an extended version of the scaffold on the basis of this observation. The resultant compound, 2,6‐bis‐(2‐carboxyphenylamino)benzoate, is a 40‐fold more potent inhibitor of the enzyme than the original hit and provides direction for further structure‐based drug design.