Christopher P. Gordon

Inhibition of dynamin mediated endocytosis by the dynoles: synthesis and functional activity of a family of indoles

Screening identified two bisindolylmaleimides as 100 microM inhibitors of the GTPase activity of dynamin I. Focused library approaches allowed development of indole-based dynamin inhibitors called dynoles. 100-Fold in vitro enhancement of potency was noted with the best inhibitor, 2-cyano-3-(1-(2-(dimethylamino)ethyl)-1H-indol-3-yl)-N-octylacrylamide (dynole 34-2), a 1.3 +/- 0.3 microM dynamin I inhibitor. Dynole 34-2 potently inhibited receptor mediated endocytosis (RME) internalization of Texas red-transferrin. The rank order of potency for a variety of dynole analogues on RME in U2OS cells matched their rank order for dynamin inhibition, suggesting that the mechanism of inhibition is via dynamin. Dynoles are the most active dynamin I inhibitors reported for in vitro or RME evaluations. Dynole 34-2 is 15-fold more active than dynasore against dynamin I and 6-fold more active against dynamin mediated RME (IC(50) approximately 15 microM; RME IC(50) approximately 80 microM). The dynoles represent a new series of tools to better probe endocytosis and dynamin-mediated trafficking events in a variety of cells.

Attenuating Staphylococcus aureus virulence gene regulation: a medicinal chemistry perspective.

Virulence gene expression in Staphylococcus aureus is tightly regulated by intricate networks of transcriptional regulators and two-component signal transduction systems. There is now an emerging body of evidence to suggest that the blockade of S. aureus virulence gene expression significantly attenuates infection in experimental models. In this Perspective, we will provide insights into medicinal chemistry strategies for the development of chemical reagents that have the capacity to inhibit staphylococcal virulence expression. These reagents can be broadly grouped into four categories: (1) competitive inhibitors of the accessory gene regulator (agr) quorum sensing system, (2) inhibitors of AgrA–DNA interactions, (3) RNAIII transcription inhibitors, and (4) inhibitors of the SarA family of transcriptional regulators. We discuss the potential of specific examples of antivirulence agents for the management and treatment of staphylococcal infections.

Aryl Nitro Reduction with Iron Powder or Stannous Chloride under Ultrasonic Irradiation

Abstract The selective reduction of aryl nitro compounds in the presence of sensitive functionalities, including halide, carbonyl, nitrile, and ester substituents, under ultrasonic irradiation at 35 kHz is reported in yields of 39–98%. Iron powder proved superior to stannous chloride with high tolerance of sensitive functional groups and high yields of the desired aryl amines in relatively short reaction times. Simple experimental procedure and purification also make the iron reduction of aryl nitro compounds advantageous over other methods of reduction.

Defining the Mechanisms by Which the Reactive Oxygen Species By-Product, 4-Hydroxynonenal, Affects Human Sperm Cell Function

ABSTRACT Lipid peroxidation products such as the naturally occurring aldehyde 4-hydroxynonenal (4-HNE) are known to be cytotoxic toward different cell types, including spermatozoa. In order to understand this at the molecular level, we have employed a proteomic approach to characterize direct 4-HNE adducts on human spermatozoa. Several proteins were identified to be of particular interest, including aldehyde labeling of histone methyltransferase and dynein heavy chain. In addition, we found that 4-HNE bound to part of the activation segment, cysteine residue 199, of protein kinase A (PKA). Interestingly, at low levels, addition of 4-HNE had a stimulatory effect on PKA. However, this did not correlate to increased phosphotyrosine levels during capacitation. This data explains the link between reactive oxygen species and sperm toxicity. Given that epigenetic regulation is likely affected in oxidative-stressed spermatozoa, this data show that spermatozoa appear to shut down under these conditions before reaching the egg.

Norcantharidin analogues: Synthesis, anticancer activity and protein phosphatase 1 and 2A inhibition

Cantharidin (1) and its derivatives are of significant interest as serine/threonine protein phosphatase 1 and 2A inhibitors. Additionally, compounds of this type have displayed growth inhibition of various tumour cell lines. To further explore both of these inhibition pathways, a number of amide–acid norcantharidin analogues (15–26) were prepared. Compounds 23 and 24, containing two carboxylic acid residues, showed good PP1 and PP2A activity, with IC50 values of ∼15 and ∼3 μm, respectively. Substituted aromatic amide analogues 45, 48, 49, 52, 53, and 54 also displayed good PP1 and PP2A inhibition, with IC50 values in the range of 15–10 μM (PP1) and 11–5 μM (PP2A). However, bulky ortho substituents on the aromatic ring caused the aromatic ring to be skewed from the NCO planarity, leading to a decrease in PP1 and PP2A inhibition. A number of analogues, 20, 22, 25 and 46, showed excellent tumour growth inhibition, with 46 in particular being more potent than the lead, norcantharidin 2.

The Dynamin Inhibitors MiTMAB and OcTMAB Induce Cytokinesis Failure and Inhibit Cell Proliferation in Human Cancer Cells

The endocytic protein dynamin II (dynII) participates in cell cycle progression and has roles in centrosome cohesion and cytokinesis. We have described a series of small-molecule inhibitors of dynamin [myristyl trimethyl ammonium bromides (MiTMAB)] that competitively interfere with the ability of dynamin to bind phospholipids and prevent receptor-mediated endocytosis. We now report that dynII functions specifically during the abscission phase of cytokinesis and that MiTMABs exclusively block this step in the cell cycle. Cells treated with MiTMABs (MiTMAB and octadecyltrimethyl ammonium bromide) and dyn-depleted cells remain connected via an intracellular bridge for a prolonged period with an intact midbody ring before membrane regression and binucleate formation. MiTMABs are the first compounds reported to exclusively block cytokinesis without affecting progression through any other stage of the cell cycle. Thus, MiTMABs represent a new class of antimitotic compounds. We show that MiTMABs are potent inhibitors of cancer cell growth and have minimal effect on nontumorigenic fibroblast cells. Thus, MiTMABs have toxicity and antiproliferative properties that preferentially target cancer cells. This suggests that dynII may be a novel target for pharmacologic intervention for the treatment of cancer. Mol Cancer Ther; 9(7); 1995–2006. ©2010 AACR.

Inhibition of dynamin by dynole 34-2 induces cell death following cytokinesis failure in cancer cells

Inhibitors of mitotic proteins such as Aurora kinase and polo-like kinase have shown promise in preclinical or early clinical development for cancer treatment. We have reported that the MiTMAB class of dynamin small molecule inhibitors are new antimitotic agents with a novel mechanism of action, blocking cytokinesis. Here, we examined 5 of the most potent of a new series of dynamin GTPase inhibitors called dynoles. They all induced cytokinesis failure at the point of abscission, consistent with inhibition of dynamin while not affecting other cell cycle stages. All 5 dynoles inhibited cell proliferation (MTT and colony formation assays) in 11 cancer cell lines. The most potent GTPase inhibitor, dynole 34-2, also induced apoptosis, as revealed by cell blebbing, DNA fragmentation, and PARP cleavage. Cell death was induced specifically following cytokinesis failure, suggesting that dynole 34-2 selectively targets dividing cells. Dividing HeLa cells were more sensitive to the antiproliferative properties of all 5 dynoles compared with nondividing cells, and nontumorigenic fibroblasts were less sensitive to cell death induced by dynole 34-2. Thus, the dynoles are a second class of dynamin GTPase inhibitors, with dynole 34-2 as the lead compound, that are novel antimitotic compounds acting specifically at the abscission stage. Mol Cancer Ther; 10(9); 1553–62. ©2011 AACR.

A facile, protic ionic liquid route to N-substituted 5-hydroxy-4-methyl-3-oxoisoindoline-1-carboxamides and N-substituted 3-oxoisoindoline-4-carboxylic acids

Treatment of highly decorated bicyclo[2.2.1]heptadienes with the protic ionic liquid, TfOH:TEA effected quantitative conversion to the corresponding N-substituted 5-hydroxy-4-methyl-3-oxoisoindoline-1-carboxamides. This approach provides rapid access important chemical space for the rapid development of highly functionalised oxoisoindoline and is highly substrate tolerant.

Calcineurin activity is required for the completion of cytokinesis.

Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca2+- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca2+ influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.

Hedgehog signalling pathway inhibitors as cancer suppressing agents

The Hedgehog (Hh) signalling pathway plays a pivotal role in the spatial and temporal regulation of cell proliferation and differentiation. By controlling the correct maturation of developing tissues and ensuring attainment of the correct size, position and the presence of fully functioning cellular structures, the Hh plays a pivotal role in development. Conversely aberrant Hh signalling is involved in Gorlin syndrome, basal cell carcinoma (the most common cancer in the world), and more than one third of all human medulloblastoma cases. In all of these cases, it is believed that deregulated Hh signalling leads to increased cell proliferation and tumour formation. Inhibition of the Hedgehog signalling pathway, is a recently validated anti-cancer drug target, with vismodegib (Erivedge™), approved by the U.S. Food and Drug Administration for the treatment of adult basal cell carcinoma. In this perspective we outline the current state of Hh pathway inhibitors with a particular focus on potential limitations of upstream Hh pathway inhibition in relation to resistance mutations and crosstalk pathways. Together, these limitations indicate that inhibition of downstream components, specifically the Gli family of transcription factors, may represent a next generation approach to suppress tumours associated with aberrant Hh pathway signalling.