Structure-Based Design, Synthesis and Biological Evaluation of Bis-Tetrahydropyran Furan Acetogenin Mimics Targeting the Trypanosomatid F1 Component of ATP Synthase.

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for the severely debilitating neglected Tropical diseases of African sleeping sickness, Chagas disease and leishmaniasis, respectively. As part of our ongoing programme exploring the potential of simplified analogues of the acetogenin chamuvarinin we identified the T. brucei FoF1-ATP synthase as a target of our earlier triazole analogue series. Using computational docking studies, we hypothesised that the central triazole heterocyclic spacer could be substituted for a central 2,5-substituted furan moiety, thus diversifying the chemical framework for the generation of compounds with greater potency and/or selectivity. Here we report the design, docking, synthesis and biological evaluation of new series of trypanocidal compounds and demonstrate their on-target inhibitory effects. Furthermore, the synthesis of furans by the modular coupling of alkyne- and aldehyde-THPs to bis-THP 1,4-alkyne diols followed by ruthenium/xantphos-catalysed heterocyclisation described here represents the most complex use of this method of heterocyclisation to date.

Approaches to CNS Drug Delivery with a Focus on Transporter-Mediated Transcytosis.

Drug delivery to the central nervous system (CNS) conferred by brain barriers is a major obstacle in the development of effective neurotherapeutics. In this review, a classification of current approaches of clinical or investigational importance for the delivery of therapeutics to the CNS is presented. This classification includes the use of formulations administered systemically that can elicit transcytosis-mediated transport by interacting with transporters expressed by transvascular endothelial cells. Neurotherapeutics can also be delivered to the CNS by means of surgical intervention using specialized catheters or implantable reservoirs. Strategies for delivering drugs to the CNS have evolved tremendously during the last two decades, yet, some factors can affect the quality of data generated in preclinical investigation, which can hamper the extension of the applications of these strategies into clinically useful tools. Here, we disclose some of these factors and propose some solutions that may prove valuable at bridging the gap between preclinical findings and clinical trials.

Direct and indirect approaches to identify drug modes of action.

Phenotypic assays are becoming increasingly more common among drug discovery practices, expanding drug target diversity as lead compounds identified through such screens are not limited to known targets. While increasing diversity is beneficial to the drug discovery process and the fight against disease, the unknown modes of action of new lead compounds can hamper drug discovery as, in most cases, the process of lead compound optimization is made difficult due to the unknown nature of the target; blindly changing substituents can prove fruitless due to the inexhaustible number of potential combinations, and it is therefore desirable to rapidly identify the targets of lead compounds developed through phenotypic screening. In addition, leads identified through target-based screening often have off-target effects that contribute towards drug toxicity, and by identifying those secondary targets, the drugs can be improved. However, the identification of a leads mode of action is far from trivial and now represents a major bottleneck in the drug discovery pipeline. This review looks at some of the recent developments in the identification of drug modes of action, focusing on phenotype-based methods using metabolomics, proteomics, transcriptomics, and genomics to detect changes in phenotype in response to the presence of the drug, and affinity-based methods using modified/unmodified drug as bait to capture and identify targets. © 2017 IUBMB Life, 70(1):9-22, 2018.

The trypanosome alternative oxidase: a potential drug target?

New drugs against Trypanosoma brucei, the causative agent of Human African Trypanosomiasis, are urgently needed to replace the highly toxic and largely ineffective therapies currently used. The trypanosome alternative oxidase (TAO) is an essential and unique mitochondrial protein in these parasites and is absent from mammalian mitochondria, making it an attractive drug target. The structure and function of the protein are now well characterized, with several inhibitors reported in the literature, which show potential as clinical drug candidates. In this review, we provide an update on the functional activity and structural aspects of TAO. We then discuss TAO inhibitors reported to date, problems encountered with in vivo testing of these compounds, and discuss the future of TAO as a therapeutic target.

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase.

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-ß-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by 31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcat shows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.

Simplifying nature: Towards the design of broad spectrum kinetoplastid inhibitors, inspired by acetogenins.

The need for new treatments for the neglected tropical diseases African sleeping sickness, Chagas disease and Leishmaniasis remains urgent with the diseases widespread in tropical regions, affecting the world's very poorest. We have previously reported bis-tetrahydropyran 1,4-triazole analogues designed as mimics of the annonaceous acetogenin natural product chamuvarinin, which maintained trypanocidal activity. Building upon these studies, we here report related triazole compounds with pendant heterocycles, mimicking the original butenolide of the natural product. Analogues were active against T. brucei, with a nitrofuran compound displaying nanomolar trypanocidal activity. Several analogues also showed strong activity against T. cruzi and L. major. Importantly, select compounds gave excellent selectivity over mammalian cells with a furan-based analogue highly selective while remaining active against all three cell lines, thus representing a potential lead for a new broad spectrum kinetoplastid inhibitor.

Total synthesis, stereochemical assignment, and biological activity of chamuvarinin and structural analogues.

A highly stereocontrolled synthesis of (+)-chamuvarinin has been completed in 1.5% overall yield over 20 steps. The key fragment coupling reactions were the addition of alkyne 8 to aldehyde 7 (under Felkin-Anh control), followed by the two step activation/cyclization to close the C20-C23 2,5-cis-substituted tetrahydrofuran ring and a Julia-Kocienski olefination at C8-C9 to introduce the terminal butenolide. The inherent flexibility of our coupling strategy led to a streamlined synthesis with 17 steps in the longest sequence (2.2% overall yield), in which the key bond couplings are reversed. In addition, a series of structural analogues of chamuvarinin have been prepared and screened for activity against HeLa cancer cell lines and both the bloodstream and insect forms of Trypanosoma brucei, the parasitic agent responsible for African sleeping sickness.

Synthesis of the originally proposed structure of palmerolide C.

A stereoselective synthesis of the proposed structure of palmerolide C (1), a cytotoxic marine macrolide isolated from the Antarctic tunicate Synoicum adareanum, utilizes a convergent carbonyl-based coupling strategy to construct the C1-C24 carbon skeleton (see scheme). Compound 1 was shown to be a diastereomer of palmerolide C, and the structural revision of the natural product is proposed.

Design and Synthesis of Broad Spectrum Trypanosomatid Selective Inhibitors.

Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern that have a devastating effect on the developing world due to their burden on human and animal health. In this work, we detail the preparation of a focused library of substituted-tetrahydropyran derivatives and their evaluation as selective chemical tools for trypanosomatid inhibition and the follow-on development of photoaffinity probes capable of labeling target protein(s) in vitro. Several of these functionalized compounds maintain low micromolar activity against Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, and Leishmania donovani. In addition, we demonstrate the utility of the photoaffinity probes for target identification through preliminary cellular localization studies.

Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues.

Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photo-affinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and ß-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.

Total synthesis and stereochemical reassignment of (+)-dolastatin 19.

[reaction: see text] A revised configurational assignment for the cytotoxic marine macrolide dolastatin 19 is proposed and validated by total synthesis. Key features of the route include an asymmetric vinylogous aldol reaction to install the isolated C13 stereocenter and (E)-trisubstituted alkene, two sequential 1,4-syn boron-mediated aldol reactions, and a Mukaiyama glycosylation to append the l-rhamnose-derived pyranoside.

Development of Simplified Heterocyclic Acetogenin Analogues as Potent and Selective Trypanosoma brucei Inhibitors.

Neglected tropical diseases caused by parasitic infections are an ongoing and increasing concern. They are a burden to human and animal health, having the most devastating effect on the world's poorest countries. Building upon our previously reported triazole analogues, in this study we describe the synthesis and biological testing of other novel heterocyclic acetogenin-inspired derivatives, namely 3,5-isoxazoles, furoxans, and furazans. Several of these compounds maintain low-micromolar levels of inhibition against Trypanosoma brucei, whilst having no observable inhibitory effect on mammalian cells, leading to the possibility of novel lead compounds for selective treatment.

An internal thioester in a pathogen surface protein mediates covalent host binding.

To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a 'chemical harpoon'. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions.

Discodermolide interferes with the binding of tau protein to microtubules.

We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

1,6-asymmetric induction in boron-mediated aldol reactions: application to a practical total synthesis of (+)-discodermolide.

By relying solely on substrate-based stereocontrol, a practical total synthesis of the microtubule-stabilizing anticancer agent (+)-discodermolide has been realized. This exploits a novel aldol bond construction with 1,6-stereoinduction from the boron enolate of (Z)-enone 3 in addition to aldehyde 2. The 1,3-diol 7 is employed as a common building block for the C(1)-C(5), C(9)-C(16), and C(17)-C(24) subunits. [reaction--see text]

A quantitative evaluation of the effects of inhibitors of tubulin assembly on polymerization induced by discodermolide, epothilone B, and paclitaxel.

PURPOSE: To determine whether inhibitors of microtubule assembly inhibit polymerization induced by discodermolide and epothilone B, as well as paclitaxel, and to quantitatively measure such effects. METHODS: Inhibition was quantitated by measuring polymer formation either by turbidimetry or by centrifugation, and the amount of inhibitor required to inhibit 50% relative to an appropriate control reaction was determined. RESULTS: The inhibitory drugs evaluated were four colchicine site agents (combretastatin A-4, podophyllotoxin, nocodazole, and N-acetylcolchinol- O-methyl ether), maytansine, which competitively inhibits the binding of Catharanthus alkaloids to tubulin, halichondrin B and phomopsin A, which noncompetitively inhibit the binding of Catharanthus alkaloids to tubulin, and the depsipeptide dolastatin 15. While relative inhibitory effects were highly variable, a few broad generalizations can be made. First, assembly reactions that were either enhanced or dependent upon all three stimulatory drugs were subject to inhibition by all inhibitors. Second, the more readily the tubulin assembled, the greater the concentration of inhibitor required to inhibit polymerization. Drug IC50 values were generally lowest with no stimulatory drug and highest when discodermolide was present; IC50 values were higher as reaction temperature increased; and IC50 values were higher as the tubulin concentration increased. Third, inhibition of assembly by inhibitors of Catharanthus alkaloid binding to tubulin changed much less as a function of changes in reaction conditions than inhibition by inhibitors of colchicine binding. CONCLUSIONS: Since there was no apparent quantitative predictability of combined drug interactions with tubulin, any combination of interest must be studied in detail.

Mucoadhesive polymer films for tissue retraction in laparoscopic surgery: Ex-vivo study on their mechanical properties.

Safe and effective manipulation of soft tissue during laparoscopic procedures can be achieved by the use of mucoadhesive polymer films. A series of novel adhesive polymer films were formulated in house based on either Carbopol or Chitosan modified systems. The mechanical properties of the polymers and their adherence to bowel were evaluated using ex-vivo pig bowel immersed in 37°C water bath and connected to an Instron tensiometer. Young's modulus was 300 kPa for the Carbopol-polymer and 5 kPa for the Chitosan-polymer. The Chitosan-polymer exhibited much larger shear adhesion than its tensile adhesion: 3.4 N vs. 1.2. Both tensile and shear adhesions contributed to the large retraction force (2.6 N) obtained during l polymer-bowel retraction testing. Work of adhesion at the polymer/serosa interface, defined as the area under the force curve, was 64 mJ, which is appreciably larger than that reported with existing polymers. In conclusion, adhesive polymers can stick to the serosal side of the bowel with an adhesive force, which is sufficient to lift the bowel, providing a lower retraction stress than that caused by laparoscopic grasping which induces high localized pressures on the tissue.

Non-natural acetogenin analogues as potent Trypanosoma brucei inhibitors.

Neglected tropical diseases remain a serious global health concern. Here, a series of novel bis-tetrahydropyran 1,4-triazole analogues based on the framework of chamuvarinin, a polyketide natural product isolated from the annonaceae plant species are detailed. The analogues synthesized display low micromolar trypanocidal activities towards both bloodstream and insect forms of Trypanosoma brucei, the causative agent of African sleeping sickness, also known as Human African Trypanosomiasis (HAT). A divergent synthetic strategy was adopted for the synthesis of the key tetrahydropyran intermediates to enable rapid access to diastereochemical variation either side of the 1,4-triazole core. The resulting diastereomeric analogues displayed varying degrees of trypanocidal activity and selectivity in structure-activity relationship studies. Together, the biological potency and calculated lipophilicity values indicate that while there is room for improvement, these derivatives may represent a promising novel class of anti-HAT agents.

Magnetic retraction of bowel by intraluminal injectable cyanoacrylate-based magnetic glue.

Magnetic retraction offers advantages over physical retraction by graspers because of reduced tissue trauma. The objectives of this study are to investigate a novel method of magnetisation of bowel segments by intraluminal injection of magnetic glue and to demonstrate the feasibility of magnetic retraction of bowel with sufficient force during minimal access surgery. Following an initial materials characterisation study, selected microparticles of stainless steel (SS410- µ Ps) were mixed with chosen cyanoacrylate glue (Loctite 4014). During intraluminal injection of the magnetic glue using ex vivo porcine colonic segments, a magnetic probe placed at the injected site ensured that the SS410-µPs aggregated during glue polymerisation to form an intraluminal mucosally adherent coagulum. The magnetised colonic segments were retracted by magnetic probes (5 and 10 mm) placed external to the bowel wall. A tensiometer was used to record the retraction force. With an injected volume of 2 mL in a particle concentration of 1 g/mL, this technique produced maximal magnetic retraction forces of 2.24 ± 0.23 N and 5.11 ± 0.34 N (n = 20), with use of 5 and 10 mm probes, respectively. The results indicate that the formation of an intraluminal coagulum based on SS410- µPs and Loctite 4014 produces sufficient magnetic retraction for bowel retraction.