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.

5-Aryl-2-(naphtha-1-yl)sulfonamido-thiazol-4(5H)-ones as clathrin inhibitors.

The development of a (Z)-5-((6,8-dichloro-4-oxo-4H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (2), rhodanine-based lead that led to the Pitstop® 2 family of clathrin inhibitors is described herein. Head group substitution and bioisosteric replacement of the rhodanine core with a 2-aminothiazol-4(5H)-one scaffold eliminated off target dynamin activity. A series of N-substituents gave first phenylglycine (20, IC50 ∼ 20 µM) then phenyl (25, IC50 ∼ 7.1 µM) and 1-napthyl sulfonamide (26, Pitstop® 2 compound, IC50 ∼ 1.9 µM) analogues with good activity, validating this approach. A final library exploring the head group resulted in three analogues displaying either slight improvements or comparable activity (33, 38, and 29 with IC50 ∼ 1.4, 1.6 and 1.8 µM respectively) and nine others with IC50 < 10 µM. These results were rationalized using in silico docking studies. Docking studies predicted enhanced Pitstop® 2 family binding, not a loss of binding, within the Pistop® groove of the reported clathrin mutant invalidating recent assumptions of poor selectivity for this family of clathrin inhibitors.

1,8-Naphthalimide derivatives: new leads against dynamin I GTPase activity.

Fragment-based in silico screening against dynamin I (dynI) GTPase activity identified the 1,8-naphthalimide framework as a potential scaffold for the design of new inhibitors targeting the GTP binding pocket of dynI. Structure-based design, synthesis and subsequent optimization resulted in the development of a library of 1,8-naphthalimide derivatives, called the Naphthaladyn™ series, with compounds 23 and 29 being the most active (IC50 of 19.1 ± 0.3 and 18.5 ± 1.7 µM respectively). Compound 29 showed effective inhibition of clathrin-mediated endocytosis (IC50(CME) 66 µM). The results introduce 29 as an optimised GTP-competitive lead Naphthaladyn™ compound for the further development of naphthalimide-based dynI GTPase inhibitors.

Phenothiazine-derived antipsychotic drugs inhibit dynamin and clathrin-mediated endocytosis.

Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine-derived APDs inhibited dynamin I (dynI) in the 2-12 µm range, the most potent being trifluoperazine (IC50 2.6 ± 0.7 µm). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in-cell target in endocytosis.

Development of quinone analogues as dynamin GTPase inhibitors.

Virtual screening of the ChemDiversity and ChemBridge compound databases against dynamin I (dynI) GTPase activity identified 2,5-bis-(benzylamino)-1,4-benzoquinone 1 as a 273 ± 106 µM inhibitor. In silico lead optimization and focused library-led synthesis resulted in the development of four discrete benzoquinone/naphthoquinone based compound libraries comprising 54 compounds in total. Sixteen analogues were more potent than lead 1, with 2,5-bis-(4-hydroxyanilino)-1,4-benzoquinone (45) and 2,5-bis(4-carboxyanilino)-1,4-benzoquinone (49) the most active with IC50 values of 11.1 ± 3.6 and 10.6 ± 1.6 µM respectively. Molecular modelling suggested a number of hydrogen bonding and hydrophobic interactions were involved in stabilization of 49 within the dynI GTP binding site. Six of the most active inhibitors were evaluated for potential inhibition of clathrin-mediated endocytosis (CME). Quinone 45 was the most effective CME inhibitor with an IC50(CME) of 36 ± 16 µM.

Development of 1,8-naphthalimides as clathrin inhibitors.

We reported the first small molecule inhibitors of the interaction between the clathrin N-terminal domain (TD) and endocyctic accessory proteins (i.e., clathrin inhibition1). Initial screening of a ∼17 000 small molecule ChemBioNet library identified 1. Screening of an existing in-house propriety library identified four substituted 1,8-napthalimides as ∼80-120 µM clathrin inhibitors. Focused library development gave 3-sulfo-N-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (18, IC50 ≈ 18 µM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (26, 27, 28, 34 with IC50 values of 22, 16, 15, and 15 µM respectively) with a further four (24, 25, 32, 33) more active than 18 with IC50 values of 10, 6.9, 12, and 10 µM, respectively. Docking studies rationalized the structure-activity relationship (SAR) with the biological data. 3-Sulfo-N-benzyl-1,8-naphthalimide, potassium salt (25) with an IC50 ≈ 6.9 µM, is the most potent clathrin terminal domain-amphiphysin inhibitor reported to date.

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.

Development of second-generation indole-based dynamin GTPase inhibitors.

Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)propyl)-2-methyl-1H-indol-3-yl)-N-octylacrylamide (2) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4(5H)-one isostere (19, IC(50(dyn I)) = 7.7 µM), reduced under flow chemistry conditions (20, IC(50(dyn I)) = 5.2 µM) or replaced by a simple amine. The latter provided a basis for a high yield library of compounds via a reductive amination by flow hydrogenation. Two compounds, 24 (IC(50 (dyn I)) = 0.56 µM) and 25 (IC(50(dyn I)) = 0.76 µM), stood out. Indole 24 is nontoxic and showed increased potency against dynamin I and II in vitro and in cells (IC(50(CME)) = 1.9 µM). It also showed 4.4-fold selectivity for dynamin I. The indole 24 compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date.

The Rhodadyns, a New Class of Small Molecule Inhibitors of Dynamin GTPase Activity.

Six focused rhodanine-based libraries, 60 compounds in total, were synthesized and evaluated as potential dynamin I GTPase inhibitors. Twenty-six were more potent than the lead compound with 13 returning IC50 values ≤10 µM, making the Rhodadyn series among the most active dynamin inhibitors reported. Two analogues were highly effective at blocking receptor-mediated endocytosis: C10 and D10 with IC50(RME) = 7.0 ± 2.2 and 5.9 ± 1.0 µM, respectively. These compounds are equipotent with the best reported in-cell dynamin inhibitors.

Triphendiol (NV-196), development of a novel therapy for pancreatic cancer.

Despite incremental progress in the treatment of pancreatic adenocarcinoma, the prognosis of patients remains poor. Here, we report the preclinical studies in pancreatic cancer cells that demonstrate the efficacy of triphendiol (NV-196, a synthetic isoflavene) both as a monotherapy and as a gemcitabine sensitizer. The in-vitro effects of triphendiol on the pancreatic cancer cell lines HPAC and MIAPaCa-2 were determined using cell proliferation, flow cytometry, and western blot analysis. The antiproliferative activity of triphendiol was also investigated in two xenograft models of pancreatic cancer (HPAC and MIAPaCa-2). As a monotherapy, triphendiol-inhibited cell proliferation-induced p53-independent G2/M cell cycle arrest and activation of the intrinsic (mitochondrial) apoptosis pathway. Triphendiol-induced apoptosis was caspase independent and death receptor independent, whereas cell necrosis was caspase mediated. Using combination index analysis, we have shown that pretreatment of pancreatic cancer cells with triphendiol enhanced the cytotoxic effect of gemcitabine, the standard of care used to treat advanced pancreatic cancer. In xenograft models of pancreatic cancer, the rate of tumor proliferation on mice coadministered with triphendiol and gemcitabine was significantly reduced when compared with the corresponding tumor proliferation rates from the respective monotherapy-control and vehicle-control groups. Triphendiol was recently granted Investigational New Drug status by the US Food and Drug Administration. These data justify the commencement of clinical studies investigating the utility of combining triphendiol and gemcitabine in patients with early-stage and late-stage pancreatic cancer.

The pthaladyns: GTP competitive inhibitors of dynamin I and II GTPase derived from virtual screening.

We report the development of a homology model for the GTP binding domain of human dynamin I based on the corresponding crystal structure of Dictyostelium discoidum dynamin A. Virtual screening identified 2-[(2-biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-4-chlorobenzoic acid (1) as a approximately 170 microM potent inhibitor. Homology modeling- and focused library-led synthesis resulted in development of a series of active compounds (the "pthaladyns") with 4-chloro-2-(2-(4-(hydroxymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (29), a 4.58 +/- 0.06 microM dynamin I GTPase inhibitor. Pthaladyn-29 displays borderline selectivity for dynamin I relative to dynamin II ( approximately 5-10 fold). Only pthaladyn-23 (dynamin I IC(50) 17.4 +/- 5.8 microM) was an effective inhibitor of dynamin I mediated synaptic vesicle endocytosis in brain synaptosomes with an IC(50) of 12.9 +/- 5.9 microM. This compound was also competitive with respect to Mg(2+).GTP. Thus the pthaladyns are the first GTP competitive inhibitors of dynamin I and II GTPase and may be effective new tools for the study of neuronal endocytosis.