Computer- and NMR-aided design of small-molecule inhibitors of the Hub1 protein

By binding to the spliceosomal protein Snu66, the human ubiquitin-like protein Hub1 is a modulator of the spliceosome performance and facilitates alternative splicing. Small molecules that bind to Hub1 would be of interest to study the protein-protein interaction of Hub1/Snu66, which is linked to several human pathologies, such as hypercholesterolemia, premature aging, neurodegenerative diseases, and cancer. To identify small molecule ligands for Hub1, we used the interface analysis, peptide modeling of the Hub1/Snu66 interaction and the fragment-based NMR screening. Fragment-based NMR screening has not proven sufficient to unambiguously search for fragments that bind to the Hub1 protein. This was because the Snu66 binding pocket of Hub1 is occupied by pH-sensitive residues, making it difficult to distinguish between pH-induced NMR shifts and actual binding events. The NMR analyses were therefore verified experimentally by microscale thermophoresis and by NMR pH titration experiments. Our study found two small peptides that showed binding to Hub1. These peptides are the first small-molecule ligands reported to interact with the Hub1 protein

Discovery of inhibitory fragments that selectively target Spire2−FMN2 interaction

Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2–FMN2 but not the Spire1–FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an in-house library of 755 compounds and subsequently validated in multiple orthogonal biophysical assays, including fluorescence polarization, microscale thermophoresis, and 1H–15N HSQC nuclear magnetic resonance. Extensive structure–activity relationships combined with molecular docking followed by chemical optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chemical probes targeting the Spire2–FMN2 interaction

Biofizyczna charakterystyka molekularnego mechanizmu nukleacji aktyny przez Białko Spire i jego partnera forminę oraz odkrycie małocząsteczkowych sond chemicznych zaburzających interakcję Spire-FMN2

Cytoszkielet aktynowy jest jedną z niezwykle istotnych struktur która zapewnia komórkom kształt i zdolność do poruszania się. Nieprawidłowa dynamika cytoszkieletu aktynowego jest powodem wielu chorów, także nowotworowych. W komórkach obecne są liczne białka należące do nukleatorów aktyny, jednakich dokładny mechanizm działania nie jest do końca zrozumiały. Tworzenie zarodków aktyny jest kluczowe dla wielu procesów komórkowych, także w stanach patologicznych takich jak tworzenie przerzutów nowotworowych. Pomimo znacznego postępu jaki dokonał się w ostatnich latach w kwestii mechanizmu nukleacji aktyny, wiele istotnych aspektów tego procesu wciąż pozostaje niejasnych. Taka sytuacja dotyczy białka Spire. Nie wiadomo bowiem, czy pomimo wcześniejszych doniesień białko to rzeczywiście pełni rolę nukleatora aktyny. Według najnowszych hipotez, sama obecność domen WH2 wiążących aktynę nie jest wystarczajacym czynnikiem do tworzenia zarodków polimeryzacji (nukleacji) aktny. Prawdopodobnie białko Spire, może pełnić funkcję nukleatora aktyny jedynie w asyście innych białek. W ramach niniejszej pracy postanowiono przynajmniej częściowo odpowiedzieć na pytania dotyczące aktywności białka Spire. W pierwszej częsci projektu realizowanej z wykorzystaniem technik mikroskopii na poziomie pojedynczych częsteczek wykazano, że tandemowo połączone someny białka Spire niewykazują aktywności nukleacyjnej. W oparciu o dostępne dane zaproponowano również model przedstawiający mechanizm współdziałania białka Spire z forminami. W drugiej części projektu postanowiono zidentywikować niskocząsteczkowe związki wiążące się do białka Spire i zaburzające jego oddziaływanie z forminą FMN2. Takie cząsteczki mogłyby zostać wykorzystane jako specyficzne sondy molekularne do śledzenia roli kompleksu Spire-FMN2 na poziomie komórkowym. Wykorzystując kombinację technik obliczeniowych i biofizycznych dokonano odkrycia związków wiążących się do białka Spire. Pomimo tego, że odkryte związki wykazują słabą zdolność do oddziaływania z białkiem Spire, stanowią pierwsze związki mogące zaburzać powstawanie kompeksu pomiedzy Spire i FMN2. Podsumowując, wyniki zaprezentowane w ramach niniejszej pracy mają istotny wkład w poznawanie mechanizmu współdziałania białek Spire i FMN2. Odkrycia te mogą mieć w przyszlości wkład w rozwój dyscyplin naukowych związanych z nukleacją aktyny. Nie można również wykluczyć ich praktycznego zastosowania.Actin cytoskeleton is one of the most important structures within cells, that confers them shape and motility. Aberrant cytoskeleton dynamics underlies a broad range of human diseases, including cancer. A large repertoire of actin nucleators is present in the cells, yet their exact mechanism of action is far from well understood. Actin nucleation is essential for many cellular processes, also in those in pathological conditions, like cancer metastasis. Although in recent years we have witnessed increased understanding of the mechanism of Spire, several important aspects of its actin nucleation activity remain unclear. In essence, it is unknown whether Spire itself can serve in cells as actin nucleator. It has been postulated that the presence of WH2 domains in Spire may not be sufficient for actin nucleation and Spire, similarly to other WH2 domain containing proteins requires collaboration with other proteins. To address these important questions about the biology of Spire, within this work we employed various methods to decipher the molecular mechanism of Spire in vitro. Our goal was also to provide chemical tools that could be used for the investigation of In the first part of the project, using single-molecule techniques, we provided here strong evidence that WH2 domains of Spire are not able to nucleate actin when they are monomers. Based on this and further experiments with human counterparts of Spire and formin, we proposed a model of the collaboration of these two proteins. In the second part of the project, we sought small-molecules targeting Spire-FMN2 interface. Molecules able to bind tightly to Spire and impair its interaction with FMN2 could be used as chemical probes to study the role of the complex in cells. Here we applied a combined in silico and biophysical screening cascade and identified several compounds that bind to Spire. Although the discovered compounds appeared to be weak binders, they are first-in-class small molecules targeting Spire-FMN2 interaction and may serve as lead structures for further development of more potent inhibitors. Altogether, the results presented here should have bearings on the molecular mechanism of Spire and its collaboration with formin FMN2. Our findings may have utilities in both basic and applied sciences related to actin nucleation

Effect of selected silyl groups on the anticancer activity of 3,4-dibromo-5-hydroxy-furan-2(5H)-one derivatives

The pharmacological effects of carbon to silicon bioisosteric replacements have been widely explored in drug design and medicinal chemistry. Here, we present a systematic investigation of the impact of different silyl groups on the anticancer activity of mucobromic acid (MBA) bearing furan-2(5H)-one core. We describe a comprehensive characterization of obtained compounds with respect to their anticancer potency and selectivity towards cancer cells. All four novel compounds exert stronger antiproliferative activity than MBA. Moreover, 3b induce apoptosis in colon cancer cell lines. A detailed investigation of the mechanism of action revealed that 3b activity stems from the down-regulation of survivin and the activation of caspase-3. Furthermore, compound 3b attenuates the clonogenic potential of HCT-116 cells. Interestingly, we also found that depending on the type of the silyl group, compound selectivity towards cancer cells could be precisely controlled. Collectively, we demonstrated the utility of silyl groups for adjusting both the potency and selectivity of silicon-containing compounds. These data reveal a link between the types of silyl group and compound potency, which could have bearings for the design of novel silicon-based anticancer drugs

PD-L1 inhibitors : different classes, activities, and mechanisms of action

Targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) interaction has become an established strategy for cancer immunotherapy. Although hundreds of small-molecule, peptide, and peptidomimetic inhibitors have been proposed in recent years, only a limited number of drug candidates show good PD-1/PD-L1 blocking activity in cell-based assays. In this article, we compare representative molecules from different classes in terms of their PD-1/PD-L1 dissociation capacity measured by HTRF and in vitro bioactivity determined by the immune checkpoint blockade (ICB) co-culture assay. We point to recent discoveries that underscore important differences in the mechanisms of action of these molecules and also indicate one principal feature that needs to be considered, which is the eventual human PD-L1 specificity

Analysis tools for single-monomer measurements of self-assembly processes

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin

Hexokinase 2 inhibition and biological effects of BNBZ and its derivatives : the influence of the number and arrangement of hydroxyl groups

Hexokinase 2 (HK2), an enzyme of the sugar kinase family, plays a dual role in glucose metabolism and mediating cancer cell apoptosis, making it an attractive target for cancer therapy. While positive HK2 expression usually promotes cancer cells survival, silencing or inhibiting this enzyme has been found to improve the effectiveness of anti-cancer drugs and even result in cancer cell death. Previously, benitrobenrazide (BNBZ) was characterized as a potent HK2 inhibitor with good anti-cancer activity in mice, but the effect of its trihydroxy moiety (pyrogallol-like) on inhibitory activity and some cellular functions has not been fully understood. Therefore, the main goal of this study was to obtain the parent BNBZ (2a) and its three dihydroxy derivatives 2b–2d and to conduct additional physicochemical and biological investigations. The research hypothesis assumed that the HK2 inhibitory activity of the tested compounds depends on the number and location of hydroxyl groups in their chemical structure. Among many studies, the binding affinity to HK2 was determined and two human liver cancer cell lines, HepG2 and HUH7, were used and exposed to chemicals at various times: 24 h, 48 h and 72 h. The study showed that the modifications to the structures of the new BNBZ derivatives led to significant changes in their activities. It was also found that these compounds tend to aggregate and exhibit toxic effects. They were found to contribute to: (a) DNA damage, (b) increased ROS production, and (c) disruption of cell cycle progression. It was observed that, HepG2, occurred much more sensitive to the tested chemicals than the HUH7 cells; However, regardless of the used cell line it seems that the increase in the expression of HK2 in cancer cells compared to normal cells which have HK2 at a very low level, is a serious obstacle in anti-cancer therapy and efforts to find the effective inhibitors of this enzyme should be intensified

Synthesis and Cytotoxicity of 2,3-Enopyranosyl C-Linked Conjugates of Genistein

A series of glycoconjugates, derivatives of genistein containing a C-glycosylated carbohydrate moiety, were synthesized and their anticancer activity was tested in vitro in the human cell lines HCT 116 and DU 145. The target compounds 15–17 were synthesized by treating ω-bromoalkyl C-glycosides derived from L-rhamnal (1) with a tetrabutylammonium salt of genistein. The new, metabolically stable analogs of previously studied O-glycosidic genistein derivatives inhibited proliferation of cancer cell lines through inhibition of the cell cycle

Terphenyl-based Small-Molecule Inhibitors of Programmed Cell Death-1/Programmed Death-Ligand 1 Protein−Protein Interaction

Here, we report a novel class of potent PD-L1/PD-1 inhibitors based on the rigidified biphenyl-inspired structure – terphenyls. Using in-silico docking, we designed and later experimentally shown the efficacy of terphenyl-based scaffolds to inhibit the PD-1/PD-L1 complex formation using various biophysical and biochemical techniques. We also report a high-resolution structure of the PD-L1 complex with our most potent inhibitors allowing the identification of key interactions with PD-L1 at the molecular level. Moreover, we show the efficacy of our most potent inhibitors at activating the antitumor response using primary T-cells derived from healthy donors. This effect was not observed even for the therapeutic antibodies. This makes our compounds prominent candidates for further optimization for anti-PD-L1 cancer treatments.</p