Regulation of cerebral blood flow boosts precise brain targeting of vinpocetine-derived ionizable-lipidoid nanoparticles.

Despite advances in active drug targeting for blood-brain barrier penetration, two key challenges persist: first, attachment of a targeting ligand to the drug or drug carrier does not enhance its brain biodistribution; and second, many brain diseases are intricately linked to microcirculation disorders that significantly impede drug accumulation within brain lesions even after they cross the barrier. Inspired by the neuroprotective properties of vinpocetine, which regulates cerebral blood flow, we propose a molecular library design centered on this class of cyclic tertiary amine compounds and develop a self-enhanced brain-targeted nucleic acid delivery system. Our findings reveal that: (i) vinpocetine-derived ionizable-lipidoid nanoparticles efficiently breach the blood-brain barrier; (ii) they have high gene-loading capacity, facilitating endosomal escape and intracellular transport; (iii) their administration is safe with minimal immunogenicity even with prolonged use; and (iv) they have potent pharmacologic brain-protective activity and may synergize with treatments for brain disorders as demonstrated in male APP/PS1 mice.

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.

Structure and Synthesis of Vindolicine and Derivatives.

This article describes the reaction of vindoline with formaldehyde and trimethyl orthoformate to prepare vindolicine, tris-vindolicinyl methane and higher molecular weight homologues. The synthesis of 10-formyl vindoline as an intermediate allowed further exploration of its chemistry, in particular the reaction with acetone which yielded a symmetrical dimer, which was further reacted with vindoline to give molecules containing three and four vindoline units. These molecules were characterized by NMR and for some of them (vindolicine, 10-formyl vindoline, 10-(1'-(but-1'-en-3'-one))-vindoline) by X-ray crystallography. Depending on the substitution and on the absence of axes of symmetry, the NMR spectra displayed non-equivalent spin systems for the vindoline moieties. The dimer formed from the double condensation of 10-formyl vindoline with acetone showed cytotoxic activity in the micromolar range.

Uncovering the Mechanism of Azepino-Indole Skeleton Formation via Pictet-Spengler Reaction by Strictosidine Synthase: A Quantum Chemical Investigation.

Strictosidine synthase (STR) catalyzes the Pictet-Spengler (PS) reaction of tryptamine and secologanin to produce strictosidine. Recent studies demonstrated that the enzyme can also catalyze the reaction of non-natural substrates to form new alkaloid skeletons. For example, the PS condensation of 1H-indole-4-ethanamine with secologanin could be promoted by the STR from Rauvolfia serpentina (RsSTR) to generate a rare class of skeletons with a seven-membered ring, namely azepino-[3,4,5-cd]-indoles, which are precursors for the synthesis of new compounds displaying antimalarial activity. In the present study, the detailed reaction mechanism of RsSTR-catalyzed formation of the rare seven-membered azepino-indole skeleton through the PS reaction was revealed at the atomic level by quantum chemical calculations. The structures of the transition states and intermediates involved in the reaction pathway were optimized, and the energetics of the complete reaction were analyzed. Based on our calculation results, the most likely pathway of the enzyme-catalyzed reaction was determined, and the rate-determining step of the reaction was clarified. The mechanistic details obtained in the present study are important in understanding the promiscuous activity of RsSTR in the formation of the rare azepino-indole skeleton molecule and are also helpful in designing STR enzymes for the synthesis of other new alkaloid skeleton molecules.

Binding order and apparent binding affinity in the bisubstrate activity of strictosidine synthase.

The Rauvolfia serpentina strictosidine synthase (RsSTR) enzyme with a bisubstrate activity is central to monoterpenoid indole alkaloid (MIA) biosynthesis pathways, as it stereoselectively condenses the terpenoid and indole metabolites, secologanin and tryptamine, respectively, into strictosidine. Here, cooperativity was aimed to be deciphered by proxy with help of a non-substrate tryptamine analog (decoy compound) to allow a bisubstrate binding without reaction, facilitating an isothermal titration calorimetry (ITC)-based analysis of the effect of the presence of one substrate on the binding of the other. Tryptamine and tryptamine analog bound to RsSTR with similar binding affinities (Kd). On the contrary, ITC revealed an exothermic titration of secologanin to RsSTR but could not fully quantify it because of weak binding. Interestingly, secologanin bound to RsSTR with an apparent binding affinity (Kd,app) of 212.1 µM in the presence of the decoy compound, as opposed to a lack of binding to RsSTR alone, strongly suggesting a "tryptamine-first" mode of binding. Conversely, binding of tryptamine analog in the presence of secologanin was enhanced >3-fold. Further, molecular dynamics simulation (MDS) analyses revealed the conformational flexibility needed for such cooperativity. Our binding studies complemented with the computational analyses suggested cooperativity in the ordered bisubstrate binding to RsSTR. Therefore, understanding thermodynamics and cooperativity in the binding of substrates or ligands would help to unravel the mechanism of enzyme catalysis and ligand-receptor interactions, and would guide the redesign of enzymes for enhanced properties and the design of inhibitors against enzymes and receptors.Communicated by Ramaswamy H. Sarma.

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.

A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Pictet-Spengler Reaction for the Chemical Synthesis of Strictosidine.

Strictosidine is the common biosynthetic precursor of Monoterpene Indole Alkaloids (MIA). A practical single-step procedure to assemble strictosidine from secologanin is described via a bioinspired Pictet-Spengler reaction. Mild conditions and purification by crystallization and flash chromatography allow access to the targeted product in fair yield.

Total Synthesis of (-)-Strictosidine and Interception of Aryne Natural Product Derivatives "Strictosidyne" and "Strictosamidyne".

Monoterpene indole alkaloids are a large class of natural products derived from a single biosynthetic precursor, strictosidine. We describe a synthetic approach to strictosidine that relies on a key facially selective Diels-Alder reaction between a glucosyl-modified alkene and an enal to set the C15-C20-C21 stereotriad. DFT calculations were used to examine the origin of stereoselectivity in this key step, wherein two of 16 possible isomers are predominantly formed. These calculations suggest the presence of a glucosyl unit, also inherent in the strictosidine structure, guides diastereoselectivity, with the reactive conformation of the vinyl glycoside dienophile being controlled by an exo-anomeric effect. (-)-Strictosidine was subsequently accessed using late-stage synthetic manipulations and an enzymatic Pictet-Spengler reaction. Several new natural product analogs were also accessed, including precursors to two unusual aryne natural product derivatives termed "strictosidyne" and "strictosamidyne". These studies provide a strategy for accessing glycosylic natural products and a new platform to access monoterpene indole alkaloids and their derivatives.

The Enantioselective Synthesis of Eburnamonine, Eucophylline, and 16'-epi-Leucophyllidine.

A synthetic approach to the heterodimeric bisindole alkaloid leucophyllidine is disclosed herein. An enantioenriched lactam building block, synthesized through palladium-catalyzed asymmetric allylic alkylation, served as the precursor to both hemispheres. The eburnamonine-derived fragment was synthesized through a Bischler-Napieralski/hydrogenation approach, while the eucophylline-derived fragment was synthesized by Friedländer quinoline synthesis and two sequential C-H functionalization steps. A convergent Stille coupling and phenol-directed hydrogenation united the two monomeric fragments to afford 16'-epi-leucophyllidine in 21 steps from commercial material.

Early and Late Steps of Quinine Biosynthesis.

The enzymatic basis for quinine 1 biosynthesis was investigated. Transcriptomic data from the producing plant led to the discovery of three enzymes involved in the early and late steps of the pathway. A medium-chain alcohol dehydrogenase (CpDCS) and an esterase (CpDCE) yielded the biosynthetic intermediate dihydrocorynantheal 2 from strictosidine aglycone 3. Additionally, the discovery of an O-methyltransferase specific for 6'-hydroxycinchoninone 4 suggested the final step order to be cinchoninone 16/17 hydroxylation, methylation, and keto-reduction.

Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site.

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.

Bioinspired Early Divergent Oxidative Cyclizations toward Pleiocarpamine, Talbotine, and Strictamine.

Toward the mavacurane and akuammilane monoterpene indole alkaloids, we developed divergent oxidative couplings between the indole nucleus (at N1 or C7) and the C16-malonate of a common tricyclic model related to strictosidine according to a biosynthetic hypothesis postulated by Hesse and Schmid. These oxidative cyclizations led selectively to the formation of the N1-C16 bond of pleiocarpamine or to the C7-C16 bond of strictamine. We were then able to obtain the scaffold of talbotine.

Quaternary stereocentres via catalytic enantioconvergent nucleophilic substitution reactions of tertiary alkyl halides.

The development of efficient methods, particularly catalytic and enantioselective processes, for the construction of all-carbon quaternary stereocentres is an important (and difficult) challenge in organic synthesis due to the occurrence of this motif in a range of bioactive molecules. One conceptually straightforward and potentially versatile approach is the catalytic enantioconvergent substitution reaction of a readily available racemic tertiary alkyl electrophile by an organometallic nucleophile; however, examples of such processes are rare. Here we demonstrate that a nickel-based chiral catalyst achieves enantioconvergent couplings of a variety of tertiary electrophiles (cyclic and acyclic α-halocarbonyl compounds) with alkenylmetal nucleophiles to form quaternary stereocentres with good yield and enantioselectivity under mild conditions in the presence of a range of functional groups. These couplings, which probably proceed via a radical pathway, provide access to an array of useful families of organic compounds, including intermediates in the total synthesis of two natural products, (-)-eburnamonine and madindoline A.

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.

Enantioselective Total Synthesis of Cymoside through a Bioinspired Oxidative Cyclization of a Strictosidine Derivative.

The first total synthesis of the caged monoterpene indole alkaloid cymoside is reported. This natural product displays a unique hexacyclic-fused skeleton whose biosynthesis implies an early oxidative cyclization of strictosidine. Our approach to the furo[3,2-b]indoline framework relied on an unprecedented biomimetic sequence which started by the diastereoselective oxidation of the indole ring into a hydroxyindolenine which triggered the addition of an enol ether and was followed by the trapping of an oxocarbenium intermediate.

Triarylaminium Radical Cation Promoted Coupling of Catharanthine with Vindoline: Diastereospecific Synthesis of Anhydrovinblastine and Reaction Scope.

A new triarylaminium radical cation promoted coupling of catharanthine with vindoline is disclosed, enlisting tris(4-bromophenyl)aminium hexachlororantimonate (BAHA, 1.1 equiv) in aqueous 0.05 N HCl/trifluoroethanol (1-10:1) at room temperature (25 °C), that provides anhydrovinblastine in superb yield (85%) with complete control of the newly formed quaternary C16' stereochemistry. A definition of the scope of aromatic substrates that participate with catharanthine in the BAHA-mediated diastereoselective coupling reaction and simplified indole substrates other than catharanthine that participate in the reaction are disclosed that identify the key structural features required for participation in the reaction, providing a generalized indole functionalization reaction that bears little structural relationship to catharanthine or vindoline.

Synthesis and biological evaluation of [18F]fluorovinpocetine, a potential PET radioligand for TSPO imaging.

Despite of various PET radioligands targeting the translocator protein TSPO 18-KDa are used for the investigations of neuroinflammatory conditions associated with neurological disorders, development of new TSPO radiotracers is still an active area of the researches with a major focus on the 18F-labelled radiotracers. Here, we report the radiochemical synthesis of [18F]vinpocetine, fluorinated analogue of previously reported TSPO radioligand, [11C]vinpocetine. Radiolabeling was achieved by [18F]fluoroethylation of apovincaminic acid with [18F]fluoroethyl bromide. [18F]vinpocetine was obtained in quantities >2.7 GBq in RCY of 13% (non-decay corrected), and molar activity >60 GBq/µmol within 95 min synthesis time. Preliminary PET studies in a cynomolgus monkey and metabolite studies by HPLC demonstrated similar results by [18F]vinpocetine as for [11C]vinpocetine, including high blood-brain barrier permeability, regional uptake pattern and fast washout from the NHP brain. These results demonstrate that [18F]fluorovinpocetine warrants further evaluation as an easier accessible alternative to [11C]vinpocetine.