Small molecules that affect the p53 pathway and their potential use in the treatment of cancer

The tumor suppressor p53 was identified 35 years ago and has since then been studied extensively, but despite all efforts, no drug or therapy directly involving it has been clinically approved - yet! A lot of potential new drugs are on their way that can reactivate p53 function by various mechanisms. Even a whole new approach called cyclotherapy has been established, during which p53 is activated in normal cells to protect patients from the adverse effects of chemotherapy while tumor cells are still being killed efficiently. In this thesis, 16 drug combinations are being described in this context (paper I). Four individual p53-activating compounds, i.e. tenovin-6, leptomycin B (LMB), nutlin-3 and actinomycin D at low doses (LDactD), were used prior to the addition of each one clinically approved chemotherapeutic agent, i.e vinblastine, vinorelbine, cytosine arabinoside or gemcitabine. LDactD, which is clinically approved, showed the most promising results. Unexpectedly, we identified two compounds that can inhibit p53’s ability to induce p21, i.e. the novel SirT2 inhibitor tenovin-D3 (paper II) and the widely used histone deacetylase inhibitor (HDACi) trichostatin A (TSA) (paper III). Inhibition of p21 in tumor cells might be desirable during cancer treatment to prevent tumor cells from undergoing cell cycle arrest, which would make them more vulnerable to classic chemotherapy. On the other hand, an inhibition of cell cycle arrest in normal cells might occur, which may worsen the side effects caused by chemotherapy. However, SirT2 plays a role in neurodegenerative diseases, and hence compounds like tenovin-D3 may be of use in the treatment thereof. Furthermore, the decrease in p21 levels may be a contributing factor in the previously observed increase in efficacy during the generation of induced pluripotent stem cells upon treatment with TSA; also tenovin-D3 could be useful in this context. With the aid of a cell-based screen we identified two small molecules that can activate p53: 1) MJ05 was one of the most active hit compounds and was very selective (paper IV); it was highly cytotoxic in ARN8, especially when combined with nutlin-3, whereas it was cytostatic or had a very mild effect in other tumor cell lines and normal cells. It inhibited tumor growth in vivo, an effect that was enhanced upon co-treatment with nutlin-3. Furthermore, MJ05 selectively killed chronic myelogenous leukemia stem cells ex vivo while having milder effects in leukocyte stem cells derived from cord blood. Preliminary data strongly suggest that MJ05 acts by inhibition of pyrimidine (deoxy-) nucleotide synthesis. 2) Despite being a hit compound in our screen, MJ25 was not very potent at activating p53 (paper V). Nevertheless, its ability to inhibit thiredoxin reductase 1 (TrxR1) and its selectivity towards melanoma cell lines compared with normal cells were interesting features. We compared it with the TrxR1 inihibitor auranofin, which was very potent and selective at killing melanoma cells in cell viability assays. The insolubility of MJ25 at concentrations required for in vivo studies prevented us from testing it on xenografts in mice. Furthermore, MJ25 might not be specific for TrxR1, so the identification of additional targets could be investigated in the future. Auranofin, the other hand, has a more defined mechanism of action and is clinically approved for the treatment of rheumatoid arthritis. These traits combined with its potentially selective cytotoxic effect at low micromolar concentrations in melanoma cells may turn this compound into a potential drug candidate to be tested in patients suffering from malignant melanoma. In the final study presented in this thesis (paper VI) we tested the small molecule tenovin-6 in zebrafish embryos The compound had been described previously by our group. The original aim of this study was to investigate if the activation of p53 in an organism could affect the ability of tumor cells to disseminate. Even though tenovin-6 did not activate wild-type p53 under the conditions tested, in vivo activity of the compound was still detectable, since embryos expressing mutant p53 (M214K) displayed an increase in p53 protein levels; furthermore, the compound was lethal in a dose- and time-dependent manner, and the embryos lost most of their brown/black pigmentation. The exact mechanism behind the latter observation could not be elucidated in the course of the project. However, tyrosinase, a key enzyme in melanogenesis, was not inhibited by tenovin-6, and the combination of data obtained by others on mutated or pharmacologically inhibited vacuolar H+-ATPase (V- ATPase) and yeast mutant strains suggested that the compound may target V-ATPase

Asymmetric Bethe-Salpeter equation for pairing and condensation

The Martin-Schwinger hierarchy of correlations are reexamined and the three-particle correlations are investigated under various partial summations. Besides the known approximations of screened, ladder and maximally crossed diagrams the pair-pair correlations are considered. It is shown that the recently proposed asymmetric Bethe-Salpeter equation to avoid unphysical repeated collisions is derived as a result of the hierarchical dependencies of correlations. Exceeding the parquet approximation we show that an asymmetry appears in the selfconsistent propagators. This form is superior over the symmetric selfconsistent one since it provides the Nambu-Gorkov equations and gap equation for fermions and the Beliaev equations for bosons while from the symmetric form no gap equation results. The selfenergy diagrams which account for the subtraction of unphysical repeated collisions are derived from the pair-pair correlation in the three-particle Greenfunction. It is suggested to distinguish between two types of selfconsistency, the channel-dressed propagators and the completely dressed propagators, with the help of which the asymmetric expansion completes the Ward identity and is $\Phi$-derivable.Comment: 12 pages. 26 figure

PSR J1856+0245: Arecibo discovery of a young energetic pulsar coincident with the TeV g-RAY source HESS J1857+026

We present the discovery of the Vela-like radio pulsar J1856+0245 in the Arecibo PALFA survey. PSR J1856+0245 has a spin period of 81 ms, a characteristic age of 21 kyr, and a spin-down luminosity Ė p 4.6 x 10 36 ergs s-1. It is positionally coincident with the TeV γ-ray source HESS J1857+026, which has no other known counterparts. Young, energetic pulsars create wind nebulae, and more than a dozen pulsar wind nebulae have been associated with very high energy (100 GeV-100 TeV) γ-ray sources discovered with the HESS telescope. The γ-ray emission seen from HESS J1857+026 is potentially produced by a pulsar wind nebula powered by PSR J1856+0245; faint X-ray emission detected by ASCA at the pulsar\u27s position supports this hypothesis. The inferred γ-ray efficiency is εγ p Lγ/Ė p 3.1 % (1-10 TeV, for a distance of 9 kpc), comparable to that observed in similar associations. © 2008. The American Astronomical Society

Arecibo pulsar survey using ALPHA: Probing radio pulsar intermittency and transients

We present radio transient search algorithms, results, and statistics from the ongoing Arecibo Pulsar ALFA (PALFA) survey of the Galactic plane. We have discovered seven objects through a search for isolated dispersed pulses. All of these objects are Galactic and have measured periods between 0.4 and 4.7 s. One of the new discoveries has a duty cycle of 0.01%, smaller than that of any other radio pulsar. We discuss the impact of selection effects on the detectability and classification of intermittent sources, and compare the efficiencies of periodicity and single-pulse (SP) searches for various pulsar classes. For some cases we find that the apparent intermittency is likely to be caused by off-axis detection or a short time window that selects only a few bright pulses and favors detection with our SP algorithm. In other cases, the intermittency appears to be intrinsic to the source. No transients were found with DMs large enough to require that they originate from sources outside our Galaxy. Accounting for the on-axis gain of the ALFA system, as well as the low gain but large solid-angle coverage of far-out sidelobes, we use the results of the survey so far to place limits on the amplitudes and event rates of transients of arbitrary origin. © 2009. The American Astronomical Society. All rights reserved.

Arecibo PALFA survey and Einstein@Home: Binary pulsar discovery by volunteer computing

We report the discovery of the 20.7ms binary pulsar J1952+2630, made using the distributed computing project Einstein@Home in Pulsar ALFA survey observations with the Arecibo telescope. Follow-up observations with the Arecibo telescope confirm the binary nature of the system. We obtain a circular orbital solution with an orbital period of 9.4hr, a projected orbital radius of 2.8lt-s, and a mass function of f = 0.15 M ⊙ by analysis of spin period measurements. No evidence of orbital eccentricity is apparent; we set a 2σ upper limit e ≲ 1.7 × 10 -3 . The orbital parameters suggest a massive white dwarf companion with a minimum mass of 0.95 M ⊙ , assuming a pulsar mass of 1.4 M ⊙ . Most likely, this pulsar belongs to the rare class of intermediate-mass binary pulsars. Future timing observations will aim to determine the parameters of this system further, measure relativistic effects, and elucidate the nature of the companion star. © 2011. The American Astronomical Society. All rights reserved

Einstein@Home DISCOVERY of A PALFA MILLISECOND PULSAR in AN ECCENTRIC BINARY ORBIT

We report the discovery of the millisecond pulsar (MSP) PSR J1950+2414 (P = 4.3 ms) in a binary system with an eccentric (e = 0.08) 22 day orbit in Pulsar Arecibo L-band Feed Array survey observations with the Arecibo telescope. Its companion star has a median mass of 0.3 Mo and is most likely a white dwarf (WD). Fully recycled MSPs like this one are thought to be old neutron stars spun-up by mass transfer from a companion star. This process should circularize the orbit, as is observed for the vast majority of binary MSPs, which predominantly have orbital eccentricities e \u3c 0.001. However, four recently discovered binary MSPs have orbits with 0. 027 \u3c e \u3c 0.44; PSR J1950+2414 is the fifth such system to be discovered. The upper limits for its intrinsic spin period derivative and inferred surface magnetic field strength are comparable to those of the general MSP population. The large eccentricities are incompatible with the predictions of the standard recycling scenario: something unusual happened during their evolution. Proposed scenarios are (a) initial evolution of the pulsar in a triple system which became dynamically unstable, (b) origin in an exchange encounter in an environment with high stellar density, (c) rotationally delayed accretion-induced collapse of a super-Chandrasekhar WD, and (d) dynamical interaction of the binary with a circumbinary disk. We compare the properties of all five known eccentric MSPs with the predictions of these formation channels. Future measurements of the masses and proper motion might allow us to firmly exclude some of the proposed formation scenarios

TIMING of 29 PULSARS DISCOVERED in the PALFA SURVEY

We report on the discovery and timing observations of 29 distant long-period pulsars found in the ongoing Arecibo L-band Feed Array pulsar survey. Following discovery with the Arecibo Telescope, confirmation and timing observations of these pulsars over several years at Jodrell Bank Observatory have yielded high-precision positions and measurements of rotation and radiation properties. We have used multi-frequency data to measure the interstellar scattering properties of some of these pulsars. Most of the pulsars have properties that mirror those of the previously known pulsar population, although four show some notable characteristics. PSRs J1907+0631 and J1925+1720 are young and are associated with supernova remnants or plerionic nebulae: J1907+0631 lies close to the center of SNR G40.5-0.5, while J1925+1720 is coincident with a high-energy Fermi γ-ray source. One pulsar, J1932+1500, is in a surprisingly eccentric, 199 day binary orbit with a companion having a minimum mass of 0.33 M o. Several of the sources exhibit timing noise, and two, PSRs J0611+1436 and J1907+0631, have both suffered large glitches, but with very different post-glitch rotation properties. In particular, the rotational period of PSR J0611+1436 will not recover to its pre-glitch value for about 12 years, a far greater recovery timescale than seen following any other large glitches