Bridging the maytansine and vinca sites: Cryptophycins target ß-tubulin's T5-loop.

Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.

Probing the Mechanisms Underlying the Transport of the Vinca Alkaloids by P-glycoprotein.

The efficacy of many cancer drugs is hindered by P-glycoprotein (Pgp), a cellular pump that removes drugs from cells. To improve chemotherapy, drugs capable of evading Pgp must be developed. Despite similarities in structure, vinca alkaloids (VAs) show disparate Pgp-mediated efflux ratios. ATPase activity and binding affinity studies show at least two binding sites for the VAs: high- and low-affinity sites that stimulate and inhibit the ATPase activity rate, respectively. The affinity for ATP from the ATPase kinetics curve for vinblastine (VBL) at the high-affinity site was 2- and 9-fold higher than vinorelbine (VRL) and vincristine (VCR), respectively. Conversely, VBL had the highest Km (ATP) for the low-affinity site. The dissociation constants (KDs) determined by protein fluorescence quenching were in the order VBL < VRL< VCR. The order of the KDs was reversed at higher substrate concentrations. Acrylamide quenching of protein fluorescence indicate that the VAs, either at 10 µM or 150 µM, predominantly maintain Pgp in an open-outward conformation. When 3.2 mM AMPPNP was present, 10 µM of either VBL, VRL, or VCR cause Pgp to shift to an open-outward conformation, while 150 µM of the VAs shifted the conformation of Pgp to an intermediate orientation, between opened inward and open-outward. However, the conformational shift induced by saturating AMPPNP and VCR condition was less than either VBL or VRL in the presence of AMPPNP. At 150 µM, atomic force microscopy (AFM) revealed that the VAs shift Pgp population to a predominantly open-inward conformation. Additionally, STDD NMR studies revealed comparable groups in VBL, VRL, and VCR are in contact with the protein during binding. Our results, when coupled with VAs-microtubule structure-activity relationship studies, could lay the foundation for developing next-generation VAs that are effective as anti-tumor agents. A model that illustrates the intricate process of Pgp-mediated transport of the VAs is presented.

Proxy-approach in understanding the bisubstrate activity of strictosidine synthases.

Besides tryptamine (1) and secologanin (2), non-cognate substrates also undergo a Pictet-Spengler reaction (PSR) catalyzed by strictosidine synthases (STR) with differing catalytic properties. We characterized the bisubstrate binding aspect of catalysis - order, affinity, and cooperativity - with STR orthologs from Rauvolfia serpentina (RsSTR) and Ophiorrhiza pumila (OpSTR) by an isothermal titration calorimetry (ITC) based 'proxy approach' that employed a non-reactive tryptamine analog (m1) to capture its inert ternary complexes with STRs and (2). ITC studies with OpSTR and (2) revealed 'tryptamine-first' cooperative binding with (1) and a simultaneous cooperative binding with (m1). Binding cooperativity among (m1) and (2) towards OpSTR was higher than RsSTR. Crystallographic study of RsSTR-(m1) complex helped to understand the unreactive binding of (m1) in terms of orientation and interactions in the RsSTR pocket. PSR with (m1) was revealed to be energetically unfeasible by the density functional theory (DFT) scans of the first hydrogen abstraction by RsSTR. The effect of pH on the bisubstrate binding to OpSTR was deciphered by molecular dynamics simulations (MDS), which also provided a molecular basis for the stability of complex of OpSTR with (m1) and (2). Therefore, we investigated STRs from a substrate binding perspective to inform drug-design and rational enzyme engineering efforts.

UPLC-ESI-QTOF-MS assisted targeted metabolomics to study the enrichment of vinca alkaloids and related metabolites in Catharanthus roseus plants grown under controlled LED environment.

Enrichment of pharmaceutically important vinca alkaloids, vinblastine and vincristine, in the leaves of Madagascar periwinkle (Catharanthus roseus) plants through different pre- or postharvest treatments or cultivation conditions, e.g., exposing the plants to UV-irradiation, has been in focus for decades. Controlled LED environment in the visible light range offers the possibility of monitoring the changes in the concentration of metabolites in the vinca alkaloid-related pathway without involving UV-related abiotic stress. In the frame of our targeted metabolomics approach, 64 vinca alkaloids and metabolites were screened with the help of a UPLC-ESI-QTOF-MS instrumental setup from the leaf extracts of C. roseus plants grown in chambers under control (medium light), low light, and high blue / high red/ high far-red conditions. Out of the 14 metabolites that could be assigned either unambiguously with authentic standards or tentatively with high resolution mass spectrometry-based methods, all three dimer vinca alkaloids, that is, 3',4'-anhydrovinblastine, vinblastine and vincristine showed an at least nine-fold enrichment under high blue irradiation when compared with the control conditions: final concentrations of 961 mg kg-1 dry weight, 33.8 mg kg-1 dry weight, and 11.7 mg kg-1 dry weight could be achieved, respectively. As supported by multivariate statistical analysis, the key metabolites of the vinca alkaloid pathway were highly represented among the metabolites that were specifically stimulated by high blue light application.

Gene coexpression networks allow the discovery of two strictosidine synthases underlying monoterpene indole alkaloid biosynthesis in Uncaria rhynchophylla.

Plant-derived monoterpene indole alkaloids (MIAs) from Uncaria rhynchophylla (UR) have huge medicinal properties in treating Alzheimer's disease, Parkinson's disease, and depression. Although many bioactive UR-MIA products have been isolated as drugs, their biosynthetic pathway remains largely unexplored. In this study, untargeted metabolome identified 79 MIA features in UR tissues (leaf, branch stem, hook stem, and stem), of which 30 MIAs were differentially accumulated among different tissues. Short time series expression analysis captured 58 pathway genes and 12 hub regulators responsible for UR-MIA biosynthesis and regulation, which were strong links with main UR-MIA features. Coexpression networks further pointed to two strictosidine synthases (UrSTR1/5) that were coregulated with multiple MIA-related genes and highly correlated with UR-MIA features (r > 0.7, P < 0.005). Both UrSTR1/5 catalyzed the formation of strictosidine with tryptamine and secologanin as substrates, highlighting the importance of key residues (UrSTR1: Glu309, Tyr155; UrSTR5: Glu295, Tyr141). Further, overexpression of UrSTR1/5 in UR hairy roots constitutively increased the biosynthesis of bioactive UR-MIAs (rhynchophylline, isorhynchophylline, corynoxeine, etc), whereas RNAi of UrSTR1/5 significantly decreased UR-MIA biosynthesis. Collectively, our work not only provides candidates for reconstituting the biosynthesis of bioactive UR-MIAs in heterologous hosts but also highlights a powerful strategy for mining natural product biosynthesis in medicinal plants.

Photopharmacology of Antimitotic Agents.

Antimitotic agents such as the clinically approved vinca alkaloids, taxanes and epothilone can arrest cell growth during interphase and are therefore among the most important drugs available for treating cancer. These agents suppress microtubule dynamics and thus interfere with intracellular transport, inhibit cell proliferation and promote cell death. Because these drugs target biological processes that are essential to all cells, they face an additional challenge when compared to most other drug classes. General toxicity can limit the applicable dose and therefore reduce therapeutic benefits. Photopharmacology aims to avoid these side-effects by introducing compounds that can be applied globally to cells in their inactive form, then be selectively induced to bioactivity in targeted cells or tissue during a defined time window. This review discusses photoswitchable analogues of antimitotic agents that have been developed by combining different photoswitchable motifs with microtubule-stabilizing or microtubule-destabilizing agents.

Enhancement of Vindoline and Catharanthine Accumulation, Antioxidant Enzymes Activities, and Gene Expression Levels in Catharanthus roseus Leaves by Chitooligosaccharides Elicitation.

Catharanthus roseus (L.) G. Don is a plant belonging to the genus Catharanthus of the Apocynaceae family. It contains more than one hundred alkaloids, of which some exhibit significant pharmacological activities. Chitooligosaccharides are the only basic aminooligosaccharides with positively charged cations in nature, which can regulate plant growth and antioxidant properties. In this study, the leaves of Catharanthus roseus were sprayed with chitooligosaccharides of different molecular weights (1 kDa, 2 kDa, 3 kDa) and different concentrations (0.01 µg/mL, 0.1 µg/mL, 1 µg/mL and 10 µg/mL). The fresh weights of its root, stem and leaf were all improved after chitooligosaccharides treatments. More importantly, the chitooligosaccharides elicitor strongly stimulated the accumulation of vindoline and catharanthine in the leaves, especially with the treatment of 0.1 µg/mL 3 kDa chitooligosaccharides, the contents of them were increased by 60.68% and 141.54%, respectively. Furthermore, as the defensive responses, antioxidant enzymes activities (catalase, glutathione reductase, ascorbate peroxidase, peroxidase and superoxide dismutase) were enhanced under chitooligosaccharides treatments. To further elucidate the underlying mechanism, qRT-PCR was used to investigate the genes expression levels of secologanin synthase (SLS), strictosidine synthase (STR), strictosidine glucosidase (SGD), tabersonine 16-hydroxylase (T16H), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT), peroxidase 1 (PRX1) and octadecanoid-responsive Catharanthus AP2-domain protein 3 (ORCA3). All the genes were significantly up-regulated after chitooligosaccharides treatments, and the transcription abundance of ORCA3, SLS, STR, DAT and PRX1 reached a maximal level with 0.1 µg/mL 3 kDa chitooligosaccharides treatment. All these results suggest that spraying Catharanthus roseus leaves with chitooligosaccharides, especially 0.1 µg/mL of 3 kDa chitooligosaccharides, may effectively improve the pharmaceutical value of Catharanthus roseus.

Alternative splicing creates a pseudo-strictosidine ß-d-glucosidase modulating alkaloid synthesis in Catharanthus roseus.

Deglycosylation is a key step in the activation of specialized metabolites involved in plant defense mechanisms. This reaction is notably catalyzed by ß-glucosidases of the glycosyl hydrolase 1 (GH1) family such as strictosidine ß-d-glucosidase (SGD) from Catharanthus roseus. SGD catalyzes the deglycosylation of strictosidine, forming a highly reactive aglycone involved in the synthesis of cytotoxic monoterpene indole alkaloids (MIAs) and in the crosslinking of aggressor proteins. By exploring C. roseus transcriptomic resources, we identified an alternative splicing event of the SGD gene leading to the formation of a shorter isoform of this enzyme (shSGD) that lacks the last 71-residues and whose transcript ratio with SGD ranges from 1.7% up to 42.8%, depending on organs and conditions. Whereas it completely lacks ß-glucosidase activity, shSGD interacts with SGD and causes the disruption of SGD multimers. Such disorganization drastically inhibits SGD activity and impacts downstream MIA synthesis. In addition, shSGD disrupts the metabolic channeling of downstream biosynthetic steps by hampering the recruitment of tetrahydroalstonine synthase in cell nuclei. shSGD thus corresponds to a pseudo-enzyme acting as a regulator of MIA biosynthesis. These data shed light on a peculiar control mechanism of ß-glucosidase multimerization, an organization common to many defensive GH1 members.

Visualizing vinca alkaloids in the petal of Catharanthus roseus using functionalized titanium oxide nanowire substrate for surface-assisted laser desorption/ionization imaging mass spectrometry.

Imaging mass spectrometry (IMS) is a powerful technique that enables analysis of various molecular species at a high spatial resolution with low detection limits. In contrast to the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) approach, surface-assisted laser desorption/ionization (SALDI) can be more effective in the detection of small molecules due to the absence of interfering background signals in low m/z ranges. We developed a functionalized TiO2 nanowire as a solid substrate for IMS of low-molecular-weight species in plant tissues. We prepared TiO2 nanowires using an inexpensive modified hydrothermal process and subsequently functionalized them chemically with various silane analogs to overcome the problem of superhydrophilicity of the substrate. Chemical modification changed the selectivity of imprinting of samples deposited on the substrate surface and thus improved the detection limits. The substrate was applied to image distribution of the metabolites in very fragile specimens such as the petal of Catharanthus roseus. We observed that the metabolites are distributed heterogeneously in the petal, which is consistent with previous results reported for the C. roseus plant leaf and stem. The intermediates corresponding to the biosynthesis pathway of some vinca alkaloids were clearly shown in the petal. We also performed profiling of petals from five different cultivars of C. roseus plant. We verified the semi-quantitative capabilities of the imprinting/imaging approach by comparing results using the LC-MS analysis of the plant extracts. This suggested that the functionalized TiO2 nanowire substrate-based SALDI is a powerful technique complementary to MALDI-MS.

Vinpocetine loaded ultradeformable liposomes as fast dissolving microneedle patch: Tackling treatment challenges of dementia.

Vinpocetine (VPN) displays poor bioavailability (~7%) and short half-life (2-3 h) justifying the frequent dosing requirement of currently marketed oral tablets (thrice daily) and thus, posing a great challenge to patient compliance. Present work envisaged to achieve an infusion like delivery through transdermal route so as to tackle aforesaid challenges. With this aim, ultradeformable liposomes (UDL) incorporated fast dissolving microneedle patch (MNP) of VPN was developed and optimized for vesicle size and percent drug entrapment (critical quality attributes, CQA) utilizing the quality by design tool. Fractional factorial design followed by combined D-optimal design were applied to identify critical material attributes and obtain their statistically verified optimum levels (Phospholipon 90G, 15.17 mM; Phospholipon 90H, 4.83 mM; sodium deoxycholate, 15 mol% and Vinpocetine, 5 mol%) showing mean vesicle size of 75.65 nm and mean drug entrapment of 87.44%. An insignificant change in CQA of optimized UDL after incorporation in MNP further represented their physical compatibility with MNP components. In vitro characterization of these microneedles revealed rapid dissolution (~2 min) and good skin penetrability with around 0.684 N axial needle fracture force (ANFF). The safety was ascertained in vitro by exposing HaCaT cells to VPN UDL MNP components. A 94.27% cell viability advocated the safe nature of excipients used in formulation. Ex vivo permeation across full thickness pig ear skin revealed a steady state flux of 11.091 µg/cm2/h via VPN UDL MNP with around 9-fold enhancement when compared to flux value achieved through VPN suspension. In vivo pharmacokinetic and pharmacodynamic study in Sprague Dawley rats showed a 3-fold rise in relative bioavailability and a comparable mean escape latency via UDL MNP as compared to its oral suspension. In addition, half-life of 14 h and MRT of 21 h further confirmed the controlled release behavior of UDL MNP for prolonged period of time. In nutshell, the developed fast dissolving microneedle patch of VPN showed promising results with the prospect of lowering dose as well as dosing frequency for improved patient compliance.

Synthesis and biological evaluation of Vinpocetine derivatives.

A new series of Vinpocetine derivatives were synthesized and evaluated for their inhibitory activity on PDE1A in vitro. Seven compounds with higher inhibitory activity were selected for surface plasmon resonance (SPR) binding experiments. Compared with Vinpocetine, these high potency compounds presented a higher binding affinity with PDE1A, which was consistent with inhibitory activity. After further screening, compounds 5, 7, 21, 34 and Vinpocetine were selected to examine the vasorelaxant effects on endothelium-intact rat thoracic aortic rings. The study suggested that the effects of compounds 7 and 21 were the most significant with the maximum value of 93.46 ±â€¯0.77% and 92.90 ±â€¯0.78% (n = 5) at a concentration of 100 µM respectively. Based on these studies, compounds 7 and 21 were considered for further development as hit compounds.

Placental effects and transfer of sildenafil in healthy and preeclamptic conditions.

BACKGROUND: The phosphodiesterase-5 inhibitor (PDE5) sildenafil has emerged as a promising treatment for preeclampsia (PE). However, a sildenafil trial was recently halted due to lack of effect and increased neonatal morbidity. METHODS: Ex vivo dual-sided perfusion of an isolated cotyledon and wire-myography on chorionic plate arteries were performed to study the effects of sildenafil and the non-selective PDE inhibitor vinpocetine on the response to the NO donor sodium nitroprusside (SNP) under healthy and PE conditions. Ex vivo perfusion was also used to study placental transfer of sildenafil in 6 healthy and 2 PE placentas. Furthermore, placental mRNA and protein levels of eNOS, iNOS, PDE5 and PDE1 were quantified. FINDINGS: Sildenafil and vinpocetine significantly enhanced SNP responses in chorionic plate arteries of healthy, but not PE placentas. Only sildenafil acutely decreased baseline tension in arteries of both healthy and PE placentas. At steady state, the foetal-to-maternal transfer ratio of sildenafil was 0·37 ±â€¯0·03 in healthy placentas versus 0·66 and 0·47 in the 2 PE placentas. mRNA and protein levels of PDE5, eNOS and iNOS were comparable in both groups, while PDE1 levels were lower in PE. INTERPRETATION: The absence of sildenafil-induced NO potentiation in arteries of PE placentas, combined with the non-PDE-mediated effects of sildenafil and the lack of PDE5 upregulation in PE, argue against sildenafil as the preferred drug of use in PE. Moreover, increased placental transfer of sildenafil in PE might underlie the neonatal morbidity in the STRIDER trial. FUND: This study was funded by an mRACE Erasmus MC grant.

Completion of the canonical pathway for assembly of anticancer drugs vincristine/vinblastine in Catharanthus roseus.

The important anticancer drugs, vinblastine, vincristine and analogs, are composed of the monoterpenoid indole alkaloids (MIAs), catharanthine and vindoline, found uniquely in the medicinal plant, Catharanthus roseus. While 26 genes involved in the assembly of these two MIAs are known, two key reactions have eluded characterization to complete the documentation of the vinblastine pathway in this plant species. The assembly of these dimeric MIAs requires O-acetylstemmadenine oxidase (ASO) and a dual function geissoschizine synthase (GS) that reduces cathenamine to form geissoschizine, and that also reduces the ASO product to form a common intermediate for subsequent conversion by four separate hydrolases to catharanthine, tabersonine or vincadifformine, respectively. The in planta role of ASO is supported by identifying a single amino acid-substituted ASO mutant with very low enzyme activity and by virus-induced gene silencing of ASO to produce plants that accumulate O-acetylstemmadenine rather than catharanthine and vindoline found in wild-type (WT) plants. The in planta role of GS is supported by showing that a low GS-expressing mutant accumulating lower levels of catharanthine and vindoline also displays significantly lower tabersonine-forming activity in coupled enzyme assays than in the WT background. Gene expression analyses demonstrate that both ASO and GS are highly enriched in the leaf epidermis where the pathways for catharanthine and tabersonine biosynthesis are expressed. The full elucidation of this canonical pathway enables synthetic biology approaches for manufacturing a broad range of MIAs, including these dimers used in cancer treatment.

Stepwise response of MeJA-induced genes and pathways in leaves of C. roseus.

Catharanthus roseus is a perennial herb known for the production of important terpenoid indole alkaloids (TIAs) in addition to a variety of phenolic compounds. The goal of the present work was to detect the prolonged effects of MeJA (6 uM) treatment across time (up to 24 days) in order to detect the stepwise response of MeJA-induced genes and pathways in leaves of C. rouses. Prolonged exposure of plants to MeJA (6 uM) treatment for different time points (6, 12 and 24 days) indicated that genes in the indole alkaloid biosynthesis pathway and upstream pathways were triggered earlier (e.g., 6 days) than those in the anthocyanin biosynthesis pathway and its upstream pathways (e.g., 12 days). Three enzymes, e.g., T16H, OMT, and D4H, in the six-step vindoline biosynthesis and two enzymes, e.g., TDC and STR, acting consecutively in the conversion of tryptophan to strictosidine, were activated after 6 days of MeJA treatment. Two other key enzymes, e.g., TRP and CYP72A1, acting concurrently upstream of the TIA biosynthesis pathway were upregulated after 6 days. The genes encoding TDC and STR might concurrently act as a master switch of the TIA pathway towards the production of the indole alkaloids. On the other hand, we speculate that the gene encoding PAL enzyme also acts as the master switch of phenylpropanoid biosynthesis and the downstream flavonoid biosynthesis and anthocyanin biosynthesis pathways towards the production of several phenolic compounds. PAL and the downstream enzymes were activated 12 days after treatment. Cluster analysis confirmed the concordant activities of the flower- and silique-specific bHLH25 transcription factor and the key enzyme in the TIA biosynthesis pathway, e.g., STR. Due to the stepwise response of the two sets of pathways, we speculate that enzymes activated earlier likely make TIA biosynthesis pathway a more favourable target in C. roseus than anthocyanin biosynthesis pathway.

Solution of the multistep pathway for assembly of corynanthean, strychnos, iboga, and aspidosperma monoterpenoid indole alkaloids from 19E-geissoschizine.

Monoterpenoid indole alkaloids (MIAs) possess a diversity of alkaloid skeletons whose biosynthesis is poorly understood. A bioinformatic search of candidate genes, combined with their virus-induced gene silencing, targeted MIA profiling and in vitro/in vivo pathway reconstitution identified and functionally characterized six genes as well as a seventh enzyme reaction required for the conversion of 19E-geissoschizine to tabersonine and catharanthine. The involvement of pathway intermediates in the formation of four MIA skeletons is described, and the role of stemmadenine-O-acetylation in providing necessary reactive substrates for the formation of iboga and aspidosperma MIAs is described. The results enable the assembly of complex dimeric MIAs used in cancer chemotherapy and open the way to production of many other biologically active MIAs that are not easily available from nature.

A tabersonine 3-reductase Catharanthus roseus mutant accumulates vindoline pathway intermediates.

MAIN CONCLUSION: Monoterpenoid indole alkaloids (MIAs) have remarkable biological properties that have led to their medical uses for a variety of human diseases. Mutagenesis has been used to generate plants with new alkaloid profiles and a useful screen for rapid comparison of MIA profiles is described. The MIA mutants identified are useful for investigating MIA biosynthesis and for targeted production of these specialised metabolites. The Madagascar periwinkle (Catharanthus roseus) is the sole source of the dimeric anticancer monoterpenoid indole alkaloids (MIAs), 3',4'-anhydrovinblastine and derivatives, which are formed via the coupling of the MIAs, catharanthine and vindoline. While intense efforts to identify parts of the complex pathways involved in the assembly of these dimers have been successful, our understanding of MIA biochemistry in C. roseus remains limited. A simple thin layer chromatography screen of 4000 ethyl methanesulfonate-metagenized M2 plants is described to identify mutant lines with altered MIA profiles. One mutant (M2-1865) accumulated reduced levels of vindoline inside the leaves in favour of high levels of tabersonine-2,3-epoxide and 16-methoxytabersonine-2,3-epoxide on the leaf surface. This MIA profile suggested that changes in tabersonine 3-reductase (T3R) activity might be responsible for the observed phenotype. Molecular cloning of mutant and wild type T3R revealed two nucleotide substitutions at cytosine residues 565 (CAT to TAT) and 903 (ACC to ACA) in the mutant corresponding to substitution (H189Y) and silent (T305T) amino acid mutations, respectively, in the protein. The single amino acid substitution in the mutant T3R protein diminished the biochemical activity of T3R by 95% that explained the reason for the low vindoline phenotype of the mutant. This phenotype was recessive and exhibited standard Mendelian single-gene inheritance. The stable formation and accumulation of epoxides in the M2-1865 mutant provides a dependable biological source of these two MIAs.

New insights into Vinca alkaloids resistance mechanism and circumvention in lung cancer.

Nowadays, lung cancer, as a health problem in worldwide, has high mortality both in men and women. Despite advances in diagnosis and surgical techniques of lung cancer in recent decades, chemotherapy is still a fundamentally and extensively useful strategy. Vinca alkaloids are a class of important and widely used drugs in the treatment of lung cancer, targeting on the Vinca binding site at the exterior of microtubule plus ends. Either intrinsic or acquired resistance to chemotherapy of Vinca alkaloids has been a major obstacle to the treatment of lung cancer, which arose great interests in studies of understanding and overcoming resistance. In this review, we focused on the application and resistance mechanisms of the Vinca alkaloids such as vinblastine, vincristine, vinorelbine and vinflunine in lung cancer. We reviewed characteristic resistance mechanisms in lung cancer including over-expression of ATP-binding cassette (ABC) transporters P-glycoprotein and structural, functional or expression alterations of ß-tubulin (ßII, ßIII, ßIV) which may devote to the development of acquired resistance to the Vinca alkaloids; multidrug-resistance proteins (MRP1, MRP2, MRP3) and RLIP76 protein have also been identified that probably play a significant role in intrinsic resistance. Lung resistance-related protein (LRP) is contributed to lung cancer therapy resistance, but is not deal with the Vinca alkaloids resistance in lung cancer. Understanding the principle of the Vinca alkaloids in clinical application and mechanisms of drug resistance will support individualized lung cancer therapy and improve future therapies.

A three enzyme system to generate the Strychnos alkaloid scaffold from a central biosynthetic intermediate.

Monoterpene indole alkaloids comprise a diverse family of over 2000 plant-produced natural products. This pathway provides an outstanding example of how nature creates chemical diversity from a single precursor, in this case from the intermediate strictosidine. The enzymes that elicit these seemingly disparate products from strictosidine have hitherto been elusive. Here we show that the concerted action of two enzymes commonly involved in natural product metabolism-an alcohol dehydrogenase and a cytochrome P450-produces unexpected rearrangements in strictosidine when assayed simultaneously. The tetrahydro-ß-carboline of strictosidine aglycone is converted into akuammicine, a Strychnos alkaloid, an elusive biosynthetic transformation that has been investigated for decades. Importantly, akuammicine arises from deformylation of preakuammicine, which is the central biosynthetic precursor for the anti-cancer agents vinblastine and vincristine, as well as other biologically active compounds. This discovery of how these enzymes can function in combination opens a gateway into a rich family of natural products.The biosynthetic pathway of preakuammicine, a monoterpene precursor of the anti-cancer agent vinblastine, has remained largely unexplored. Here, the authors provide transcriptomic and biochemical data to identify two enzymes that, in tandem, convert strictosidine to akuammicine, the stable shunt product of preakuammicine.

Triazolopyrimidines Are Microtubule-Stabilizing Agents that Bind the Vinca Inhibitor Site of Tubulin.

Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.