Prolonged idasanutlin (RG7388) treatment leads to the generation of p53-mutated cells

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations

CA-170 : a potent small-molecule PD-L1 inhibitor or not?

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins – important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 – a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57

Design, Synthesis, and Biological Evaluation of 2-Hydroxy-4-phenylthiophene-3-carbonitrile as PD-L1 Antagonist and Its Comparison to Available Small Molecular PD-L1 Inhibitors

In search of a potent small molecular PD-L1 inhibitor, we designed and synthesized a compound based on a 2-hydroxy-4-phenylthiophene-3-carbonitrile moiety. Ligand's performance was tested in vitro and compared side-by-side with a known PD-L1 antagonist with a proven bioactivity BMS1166. Subsequently, we modified both compounds to allow 18F labeling that could be used for PET imaging. Radiolabeling, which is used in drug development and diagnosis, was applied to investigate the properties of those ligands and test them against tissue sections with diverse expression levels of PD-L1. We confirmed biological activity toward hPD-L1 for this inhibitor, comparable with BMS1166, while holding enhanced pharmacological properties. </p

Characterization of human osteosarcoma U-2 OS cells with acquired resistance to RG7388 – a small-molecule inhibitor of p53-MDM2 interaction

Celem niniejszej pracy było scharakteryzowanie wyprowadzonych uprzednio populacji komórek linii U-2 OS (kostniakomięsak) z nabytą opornością na związek RG7388. Jest to drobnocząsteczkowy inhibitor oddziaływania p53-MDM2 i zarazem jeden z najbardziej obiecujących potencjalnych leków przeciwnowotworowych, spośród wszystkich syntetycznych antagonistów białka MDM2. Ogromny potencjał RG7388 wykazano w eksperymentach prowadzonych na ludzkich nowotworowych liniach komórkowych oraz mysich modelach ksenograficznych. Związek ten poddawany jest obecnie testom klinicznym, zarówno jako samodzielny lek, jak i w terapiach kombinowanych. Jak dotąd, nie przeprowadzono jednak żadnych badań w kierunku zweryfikowania zjawiska nabywania oporności przez komórki nowotworowe na terapię z wykorzystaniem RG7388. W prowadzonych w naszym zespole doświadczeniach wykazano natomiast, że zjawisko to ma miejsce, co umożliwiło wyprowadzenie kilku populacji komórek zdolnych do wzrostu w obecności testowanego związku.W ramach przedstawionej pracy, z wykorzystaniem testu MTT wykazano, że komórki wyprowadzonych populacji opornych w porównaniu do linii wyjściowej U-2 OS cechuje większa przeżywalność w hodowli w obecności potencjalnego leku. Metodą hybrydyzacji western blot zweryfikowano potencjał RG7388 do zwiększania poziomu białka p53 w komórkach. Zaobserwowano ponadto, że białko p53 aktywowane w komórkach opornych nie jest zdolne do spełniania swoich funkcji w kontekście aktywacji ekspresji p21. Wyniki analizy cytometrycznej wskazały, że skutkuje to opornością komórek nowotworowych na indukowane przez RG7388 zatrzymanie w cyklu komórkowym. W ramach prezentowanej pracy dowiedziono również, że niewrażliwość komórek linii U-2 OS na terapię z wykorzystaniem antagonisty MDM2 związana jest z nabywaniem nowych cech przez komórki podczas hodowli w obecności RG7388, a nie jest jedynie konsekwencją obecności subpopulacji komórek opornych w wyjściowej heterogenicznej populacji.Uzyskane w trakcie realizacji badań wyniki uzasadniają sens poszukiwania strategii minimalizujących zjawisko nabywania lekooporności, np. poprzez testowanie interakcji RG7388 z innymi substancjami bioaktywnymi.The aim of this study was to characterize previously derived human U-2 OS (osteosarcoma) cell populations with acquired resistance to RG7388. This compound is a small molecule inhibitor of p53-MDM2 interaction and belongs to the most promising potential anti-cancer drugs among synthetic MDM2 antagonists. The significant potential of RG7388 has been demonstrated in human tumor cell lines and mouse xenograft models. The compound is currently being tested in both clinical trials as well as in combination therapies. However, no studies have been conducted to determine the acquisition of resistance by tumor cells to treatment with RG7388. Experiments carried out in our team confirm that this phenomenon has taken place. In addition, it has led to the emergence of several populations of cells capable of growth in the presence of the compound. The MTT assay was performed to demonstrate that derived populations of cells in comparison to U-2 OS parental line exhibit greater survival in culture in the presence of a potential drug. The western blot method verified the potential of RG7388 to increase p53 protein level in tumor cells. It has also been observed that p53 is not capable to activate p21 expression in resistant cells. Results of flow cytometric analysis confirmed that this phenomenon caused tumor cell resistance to RG7388-induced cell cycle arrest. The study demonstrated also that the insensitivity of U-2 OS cell lines to MDM2 antagonist therapy is related to acquisition of new features by cells during culture in the presence of RG7388 and that the observed insensitivity in not a consequence of presence of drug-resistant subpopulations in a heterogeneous parental population.Obtained results justify the need for focusing on strategies minimizing the phenomenon of drug resistance, e.g. by verification of interactions of RG7388 with other bioactive substances

Characterization of proteolytic properties of Staphylococcus pseudintermedius 222

Peptydazy wydzielane do podłoża przez różne szczepy bakterii pełnią biologiczną rolę czynników wirulencji oraz egzotoksyn. Znajdują także zastosowanie praktyczne w wielu dziedzinach biochemii i biotechnologii oraz do celów biomedycznych jako leki, szczepionki i środki grzybobójcze. Celem niniejszej pracy była próba zidentyfikowania enzymu proteolitycznego wydzielanego do podłoża przez nowy szczep gronkowca Staphylococcus pseudintermedius 222. Szczep ten jest oportunistycznym patogenem zwierzęcym, wyizolowanym z patologicznych zmian skórnych psów. Przeprowadzono oznaczenia zymograficzne na różnych substratach białkowych oraz testy aktywności proteolitycznej ze substratem białkowym znakowanym. Uzyskane wyniki pozwoliły na potwierdzenie aktywności proteolitycznej białek wydzielanych przez badane bakterie. Oszacowano także masę cząsteczkową i dokonano prób określenia sekwencji N - końca badanego enzymu.Peptidases secreted into the medium by many bacteria functions as virulence factors and exotoxins. They have also practical applications in biochemistry, biotechnology and biomedicine as drugs, vaccines or fungicides. The aim of this study was to identify the proteolityc enzyme secreted into the medium by a new strain Staphylococcus pseudintermedius 222. Such bacterium is an opportunistic pathogen of animals and was isolated from pathological skin lesions of dogs. Performed experiments included zymography with different protein substrates as well as the assays of proteolytic activity using the labeled proteins. The results confirmed the proteolityc activity of S. pseudintermedius 222 secretions and allowed also to estimate the molecular mass of the putative enzyme as well as to assess its N–terminal sequence

Antiviral activity of novel oseltamivir derivatives against some influenza virus strains

The aim of this study was to investigate the in vitro cytotoxicity of oseltamivir derivatives and determine their activity against A/H1N1/PR/8/34 and A/H3N2/HongKong/8/68 - strains of influenza virus. Antiviral activity of these compounds was determined by using two methods. MTT staining was used to assess the viability of MDCK cells infected with influenza viruses and treated with various concentrations of drugs. In parallel, the effect of drugs on viral replication was assessed using the hemagglutination test. The most toxic compounds were: OS-64, OS-35, OS-29, OS-27 and OS-25, whereas OS-11, OS-20 and OS-23 were the least toxic ones. Statistically significant antiviral effect at a higher virus dose was shown by compounds: OS-11, OS-20, OS-27, OS-35, and OS-64. H3N2 virus was sensitive to 10-times lower concentrations of OS-11 and OS-35 than H1N1. At a lower infection dose, the antiviral activity was observed for OS-11, OS 27, OS-35 and OS-20. OS-64 turned out to be effective only at a high concentration. OS-23 showed no antiviral effect