The sulfonadyns: a class of aryl sulfonamides inhibiting dynamin I GTPase and clathrin mediated endocytosis are anti-seizure in animal models.

We show that dansylcadaverine (1) a known in-cell inhibitor of clathrin mediated endocytosis (CME), moderately inhibits dynamin I (dynI) GTPase activity (IC50 45 µM) and transferrin (Tfn) endocytosis in U2OS cells (IC50 205 µM). Synthesis gave a new class of GTP-competitive dynamin inhibitors, the Sulfonadyns™. The introduction of a terminal cinnamyl moiety greatly enhanced dynI inhibition. Rigid diamine or amide links between the dansyl and cinnamyl moieties were detrimental to dynI inhibition. Compounds with in vitro inhibition of dynI activity <10 µM were tested in-cell for inhibition of CME. These data unveiled a number of compounds, e.g. analogues 33 ((E)-N-(6-{[(3-(4-bromophenyl)-2-propen-1-yl]amino}hexyl)-5-isoquinolinesulfonamide)) and 47 ((E)-N-(3-{[3-(4-bromophenyl)-2-propen-1-yl]amino}propyl)-1-naphthalenesulfonamide)isomers that showed dyn IC50 <4 µM, IC50(CME) <30 µM and IC50(SVE) from 12-265 µM. Both analogues (33 and 47) are at least 10 times more potent that the initial lead, dansylcadaverine (1). Enzyme kinetics revealed these sulfonamide analogues as being GTP competitive inhibitors of dynI. Sulfonadyn-47, the most potent SVE inhibitor observed (IC50(SVE) = 12.3 µM), significantly increased seizure threshold in a 6 Hz mouse psychomotor seizure test at 30 (p = 0.003) and 100 mg kg-1 ip (p < 0.0001), with similar anti-seizure efficacy to the established anti-seizure medication, sodium valproate (400 mg kg-1). The Sulfonadyn™ class of drugs target dynamin and show promise as novel leads for future anti-seizure medications.

A methanol and protic ionic liquid Ugi multicomponent reaction path to cytotoxic α-phenylacetamido amides.

The Ugi four component reaction of an aldehyde, amine, isocyanide and an ethanoic acid was effected smoothly in protic ionic liquids ethylammonium nitrate (EAN) and propylammonium nitrate (PAN) to afford analogues of α-phenylacetamido amides in good to excellent isolated yields. The corresponding reactions in [BMIM][PF6] and the protic ionic liquid ethanolammonium nitrate (ETAN) failed. Microwave irradiation in EAN facilitated rapid access to three focused libraries, based on the parent isocyanide: cyclohexyl isocyanide, benzyl isocyanide and ethyl isocyanoacetate. Analysis of the structure activity relationship data suggested the presence of a bulky moiety originating from the isocyanide (cyclohexyl and benzyl) enhanced cytotoxicity. Removal of the acetylenic H-atom from the ethanoic acid moiety was detrimental to cytotoxicity. The most active analogues produced, N-(2-cyclohexylamino)-1-(4-methoxyphenyl)-2-oxoethyl-N-(3,5-dimethoxyphenyl)propiolamide, returned average GI50 values of ≤1 µM across the cancer cell lines evaluated. Combined, these data suggest that analogues of this nature are interesting potential anti-cancer development leads.

FD5180, a Novel Protein Kinase Affinity Probe, and the Effect of Bead Loading on Protein Kinase Identification.

The effects of compound loading on the identification of protein kinases (PKs) was examined using two previously reported sepharose-supported PK inhibitors (PKIs): bisindolylmaleimide X (S1) and CZC8004 (S2). Compound loadings of 0.1, 0.5, 2.5, 5, 10, 25, and 50% content and an ethanolamine-blocked control bead (no compound) were investigated. A 50% bead loading gave the highest level of PK identification for both S1 and S2, extracting 34 and 55 PKs, respectively, from a single cell lysate. Control beads allowed overall identification of 23 PKs, which we term the kinase beadome, whereas sepharose-supported sunitinib (S7; 50% loading) identified 20, 11 of which were common to the control beads. The reliability of bead pull-downs was examined in duplicate experiments using two independently synthesized batches each of S1 and S2. Bead S1 showed high similarity in the absolute numbers of PKs identified across two experiments, at 40 and 35 PKs, of which 26 were common across the two batches of beads, with 14 and 9 unique PKs identified in each experiment. The S2 beads extracted 61 and 64 PKs with 55 PKs common across the two bead batches examined. We also report on the development and use of a novel promiscuous PKI analogue, 2-[(5-chloro-2{[4-(piperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]-N-methylbenzene-sulfonamide (S15), which extracted 12 additional unique PKs over the two parent compounds from which it was designed, the combination of which identifies 160 unique PKs. S15 was based on the common pyrimidine core scaffold of S9 and S10. Thus, S15 expands the utility of kinobeads by broadening the kinome coverage for bead-based pull-down. Combining the data for all beads across 90 and 180 min liquid chromatography-mass spectrometry (LC-MS)/MS analysis identified a total of 160 unique PKs.

Pyrimidine-Based Inhibitors of Dynamin I GTPase Activity: Competitive Inhibition at the Pleckstrin Homology Domain.

The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin localization to the plasma membrane via the PH domain and implicate this mechanism in the inhibition of CME. We have used a computational approach of binding site identification, docking, and interaction energy calculations to design and synthesize a new library of aminopyrimidine analogues targeting site-2 of the pleckstrin homology (PH) domain. The optimized analogues showed low micromolar inhibition against both dynamin I (IC50 = 10.6 ± 1.3 to 1.6 ± 0.3 µM) and CME (IC50(CME) = 65.9 ± 7.7 to 3.7 ± 1.1 mM), which makes this series among the more potent inhibitors of dynamin and CME yet reported. In CME and growth inhibition cell-based assays, the data obtained was consistent with dynamin inhibition. CEREP ExpresS profiling identified off-target effects at the cholecystokinin, dopamine D2, histamine H1 and H2, melanocortin, melatonin, muscarinic M1 and M3, neurokinin, opioid KOP and serotonin receptors.

Synthesis of the Pitstop family of clathrin inhibitors.

This protocol describes the synthesis of two classes of clathrin inhibitors, Pitstop 1 and Pitstop 2, along with two inactive analogs that can be used as negative controls (Pitstop inactive controls, Pitnot-2 and Pitnot-2-100). Pitstop-induced inhibition of clathrin TD function acutely interferes with clathrin-mediated endocytosis (CME), synaptic vesicle recycling and cellular entry of HIV, whereas clathrin-independent internalization pathways and secretory traffic proceed unperturbed; these reagents can, therefore, be used to investigate clathrin function, and they have potential pharmacological applications. Pitstop 1 is synthesized in two steps: sulfonation of 1,8-naphthalic anhydride and subsequent reaction with 4-amino(methyl)aniline. Pitnot-1 results from the reaction of 4-amino(methyl)aniline with commercially available 4-sulfo-1,8-naphthalic anhydride potassium salt. Reaction of 1-naphthalene sulfonyl chloride with pseudothiohydantoin followed by condensation with 4-bromobenzaldehyde yields Pitstop 2. The synthesis of the inactive control commences with the condensation of 4-bromobenzaldehyde with the rhodanine core. Thioketone methylation and displacement with 1-napthylamine affords the target compound. Although Pitstop 1-series compounds are not cell permeable, they can be used in biochemical assays or be introduced into cells via microinjection. The Pitstop 2-series compounds are cell permeable. The synthesis of these compounds does not require specialist equipment and can be completed in 3-4 d. Microwave irradiation can be used to reduce the synthesis time. The synthesis of the Pitstop 2 family is easily adaptable to enable the synthesis of related compounds such as Pitstop 2-100 and Pitnot-2-100. The procedures are also simple, efficient and amenable to scale-up, enabling cost-effective in-house synthesis for users of these inhibitor classes.

Synthesis of Dynole 34-2, Dynole 2-24 and Dyngo 4a for investigating dynamin GTPase.

Dynamin is a large GTPase with roles in membrane fission during clathrin-mediated endocytosis, in actin dynamics and in cytokinesis. Defects in dynamin have been linked to human diseases. The synthesis of a dynamin modulator toolkit comprising two different inhibitor classes is described. The first series comprises Dynole 34-2, Dynole 2-24 and the inactive control Dynole 31-2. The Dynole compounds act on the dynamin G domain, are not GTP competitive and can be synthesized in 2-3 d. Knoevenagel condensation of 1-(3-(dimethylamino)propyl)-1H-indole-3-carbaldehyde (1) with cyanoamides (2 and 3) affords Dynole 31-2 and Dynole 34-2, respectively. Reductive amination of 1 with decylamine gives Dynole 2-24. The second series acts at an allosteric site in the G domain of dynamin and comprises Dyngo 4a and Dyngo Ø (inactive control). Both are synthesized in an overnight reaction via condensation of 3-hydroxy-2-naphthoic hydrazide with 2,4,5-trihydroxybenzaldehyde to afford Dyngo 4a, or with benzaldehyde to afford Dyngo Ø.

Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC50 ~ 15 µM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC50 = 479 µM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC50 of 5.7 and 5.8 µM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

Pyrimidyn compounds: dual-action small molecule pyrimidine-based dynamin inhibitors.

Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based "Pyrimidyn" compounds that inhibit the lipid-stimulated GTPase activity of full length dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 µM for dynamin I and 1.8 µM for dynamin II, making it among the most potent dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of dynamin. The compound competitively inhibits both GTP and phospholipid interactions with dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to dynamin, limiting both the recruitment of dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of dynamin's role in endocytosis.

Synthesis and evaluation of novel ellipticines as potential anti-cancer agents.

Drugs that inhibit DNA topoisomerase I and DNA topoisomerase II have been widely used in cancer chemotherapy. We report herein the results of a focused medicinal chemistry effort around novel ellipticinium salts which target topoisomerase I and II enzymes with improved solubility. The salts were prepared by reaction of ellipticine with the required alkyl halide and evaluated for DNA intercalation, topoisomerase inhibition and growth inhibition against 12 cancer cell lines. Results from the topoisomerase I relaxation assay indicated that all novel ellipticine derivatives behaved as intercalating agents. At a concentration of 100 µM, specific topoisomerase I inhibition was not observed. Two of the derivatives under investigation were found to fully inhibit the DNA decatenation reaction at a concentration of 100 µM, indicative of topoisomerase II inhibition. N-Alkylation of ellipticine was found to enhance the observed growth inhibition across all cell lines and induce growth inhibition comparable to that of Irinotecan (CPT-11; GI(50) 1-18 µM) and in some cell lines better than Etoposide (VP-16; GI(50) = 0.04-5.2 µM). 6-Methylellipticine was the most potent growth inhibitory compound assessed (GI(50) = 0.47-0.9 µM). N-Alkylation of 6-methylellipticine was found to reduce this response with GI(50) values in the range of 1.3-28 µM.

Investigation of the one-pot synthesis of quinolin-2-(1H)-ones and the discovery of a variation of the three-component Ugi reaction.

Rapid access to the quinolin-2-(1H)-one scaffold is afforded by a sequential 4 component Ugi­Knoevenagel condensation of an aminophenylketone, an aromatic aldehyde possessing electron donating moieties, cyanoacetic acid and an aliphatic isocyanide, in moderate to good yields (49­71%). Interestingly, when the reaction is performed using aromatic aldehydes bearing electron withdrawing moieties or isocyanides containing aromatic or ester units, a mixture of a quinolin-2-(1H)-one and an α-amino amide (Ugi three-component adduct) is afforded in varying ratios. Further when the reaction is performed utilizing a combination of an isocyanide-containing aromatic or carbonyl unit, and an aldehyde possessing an electron withdrawing functionality, the Ugi three-component adduct is exclusively afforded. In our hands this new variation of the Ugi 3CR proved to be efficient and robust affording analogues in good yields (51­70%).

Exploring DNA topoisomerase I inhibition by the benzo[c]phenanthridines fagaronine and ethoxidine using steered molecular dynamics.

The benzo[c]phenanthridines (BCPs) are a group of compounds that are believed to express their antitumor activity through the inhibition of topoisomerase I. The enzyme is crucial to cell cycle division and progression, and regulates the equilibrium between relaxed and supercoiled DNA that occurs during DNA replication. Over the years, we have prepared a number of BCPs and employed a number of biophysical techniques to explore their mechanism of action and improve their activity against this particular enzyme. The naturally occurring alkaloid fagaronine 1 and the synthetic compound ethoxidine 3 are two of the most active compounds, although their inhibitory mechanisms are different, being a poison and suppressor, respectively. We have modified the approach of steered molecular dynamics to create a torque on the intercalator to comprehensively sample the DNA binding site, and using topoisomerase I crystal structures, have proposed a model to explain the different mechanisms of action for these two BCP compounds.