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 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, IC50(dyn I) = 7.7 µM), reduced under flow chemistry conditions (20, IC50(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 (IC50 (dyn I) = 0.56 µM) and 25 (IC50(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 (IC50(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

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

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

Phenothiazine-derived antipsychotic drugs inhibit dynamin and vlathrin-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 Second-Generation Indole-Based Dynamin GTPase Inhibitors

Focused library development of our lead 2-cyano-3-(1-(3-(dimethylamino)­propyl)-2-methyl-1<i>H</i>-indol-3-yl)-<i>N</i>-octylacrylamide (<b>2</b>) confirmed the tertiary dimethylamino-propyl moiety as critical for inhibition of dynamin GTPase. The cyanoamide moiety could be replaced with a thiazole-4­(5<i>H</i>)-one isostere (<b>19</b>, IC_50(dyn I) = 7.7 μM), reduced under flow chemistry conditions (<b>20</b>, 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, <b>24</b> (IC_50 (dyn I) = 0.56 μM) and <b>25</b> (IC_50(dyn I) = 0.76 μM), stood out. Indole <b>24</b> 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 <b>24</b> compound has improved isoform selectivity and is the most active in-cell inhibitor of clathrin-mediated endocytosis reported to date

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 inhibition). Initial screening of a ∼17 000 small molecule ChemBioNet library identified <b>1</b>. 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-<i>N</i>-(4-aminobenzyl)-1,8-naphthalimide, potassium salt (<b>18</b>, IC₅₀ ≈ 18 μM). A second library targeting the 4-aminobenzyl moiety was developed, and four analogues displayed comparable activity (<b>26</b>, <b>27</b>, <b>28</b>, <b>34</b> with IC₅₀ values of 22, 16, 15, and 15 μM respectively) with a further four (<b>24</b>,<b> 25</b>,<b> 32</b><i>,</i><b> 33</b>) more active than <b>18</b> 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-<i>N</i>-benzyl-1,8-naphthalimide, potassium salt (<b>25</b>) with an IC₅₀≈ 6.9 μM, is the most potent clathrin terminal domain–amphiphysin inhibitor 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: <b>C10</b> and <b>D10</b> with IC_50(RME) = 7.0 ± 2.2 and 5.9 ± 1.0 μM, respectively. These compounds are equipotent with the best reported in-cell dynamin inhibitors

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