Identification of small-molecule elastase inhibitors as antagonists of IL-36 cytokine activation.

IL-1 family cytokines act as apical initiators of inflammation in many settings and can promote the production of a battery of inflammatory cytokines, chemokines and other inflammatory mediators in diverse cell types. IL-36α, IL-36ß and IL-36γ, which belong to the extended IL-1 family, have been implicated as key initiators of skin inflammation in psoriasis. IL-36γ is highly upregulated in lesional skin from psoriatic individuals, and heritable mutations in the natural IL-36 receptor antagonist result in a severe form of psoriasis. IL-36 family cytokines are initially expressed as inactive precursors that require proteolytic processing for activation. The neutrophil granule-derived protease elastase proteolytically processes and activates IL-36α and IL-36γ, increasing their biological activity ~ 500-fold, and also robustly activates IL-1α and IL-33 through limited proteolytic processing. Consequently, inhibitors of elastase activity may have potential as anti-inflammatory agents through antagonizing the activation of multiple IL-1 family cytokines. Using in silico screening approaches, we have identified small-molecule inhibitors of elastase that can antagonize activation of IL-36γ by the latter protease. The compounds reported herein may have utility as lead compounds for the development of inhibitors of elastase-mediated activation of IL-36 and other IL-1 family cytokines in inflammatory conditions, such as psoriasis.

Design, in silico prioritization and biological profiling of apoptosis-inducing lactams amenable by the Castagnoli-Cushman reaction.

Five lactam chemotypes amenable by the Castagnoli-Cushman reaction of imines and cyclic anhydrides have been investigated for their ability to activate p53 tumor suppressing transcription factor thus induce apoptosis in p53+ cancer cells. A virtual library of 1.07 million chemically diverse compounds based on these scaffolds was subjected to in silico screening first. The compounds displaying high docking score were visually prioritized to identify the best-fitting compounds, i. e. the ones which adequately mimic the interactions of clinical candidate inhibitor Nutlin-3a. These 38 compounds were synthesized and tested for apoptosis induction in p53+ H116 cancer cells to identify 9 potent apoptosis-inducers (two of them exceeding the activity of Nutlin-3a) which belonged to four different chemotypes. The activation of p53 involved in the proapoptotic activity observed was supported by effective induction of EGFP expression in human osteocarcinoma U2OS-pLV reporter cell line. Moreover, the two most potent apoptosis inducers displayed antiproliferative profile identical to several known advanced p53 activators: they inhibited the growth of p53+/+ HCT116 cells in much lower concentration range compared to p53-/- HCT116 cells.

Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases.

Sterile inflammation is initiated by molecules released from necrotic cells, called damage-associated molecular patterns (DAMPs). Members of the extended IL-1 cytokine family are important DAMPs, are typically only released through necrosis, and require limited proteolytic processing for activation. The IL-1 family cytokines, IL-36α, IL-36ß, and IL-36γ, are expressed as inactive precursors and have been implicated as key initiators of psoriatic-type skin inflammation. We have recently found that IL-36 family cytokines are proteolytically processed and activated by the neutrophil granule-derived proteases, elastase, and cathepsin G. Inhibitors of IL-36 processing may therefore have utility as anti-inflammatory agents through suppressing activation of the latter cytokines. We have identified peptide-based pseudosubstrates for cathepsin G and elastase, based on optimal substrate cleavage motifs, which can antagonize activation of all three IL-36 family cytokines by the latter proteases. Human psoriatic skin plaques displayed elevated IL-36ß processing activity that could be antagonized by peptide pseudosubstrates specific for cathepsin G. Thus, antagonists of neutrophil-derived proteases may have therapeutic potential for blocking activation of IL-36 family cytokines in inflammatory conditions such as psoriasis.

Amino acids as chiral derivatizing agents for antiproliferative substituted N-benzyl isoindolinones.

The absolute configurations of the diastereomers of novel amino acid ester derivatives of 2,3-substituted isoindolinones, which are known as apoptosis activators due to their ability to inhibit the MDM2-p53 PPI, were assigned using NMR and computational methods. Procedures for diastereomer separation and determining the absolute configuration were developed to perform the study. The high significance of N-benzyl fragment for the determination of the diastereomer absolute configuration by NMR methods was established; it is determined by a number of factors inherent in this fragment and the structural features of the studied substrates. Analysis of the individual isomer activity showed that the target inhibitory effect of S- and R-isoindolinone L-valinates differs by less than 20%. It can be explained by the presence of a flexible linker between the isoindolinone core and amino acid fragment, which provides the optimal arrangement of the molecule in the hydrophobic cavity of MDM2 for both isomers.

Advanced palladium free approach to the synthesis of substituted alkene oxindoles via aluminum-promoted Knoevenagel reaction.

A synthetic route for the synthesis of C24, as well as for the design of focused libraries of direct AMPK activators was developed based on a convergent strategy. The proposed scheme corresponds to the current trends in C-H bond functionalization. The use of aluminum isopropoxide for the Knoevenagel condensation of oxindole with benzophenones is a noticeable point of this work.

Proapoptotic modification of substituted isoindolinones as MDM2-p53 inhibitors.

A series of novel amino acid ester derivatives of 2,3-substituted isoindolinones was synthesized and evaluated for p53-mediated apoptotic activity. The rationale for augmentation of the target activity of 2,3-substituted isoindolinones was based on the introduction of new fragments in the structure of the inhibitor that would provide additional binding sites in the hydrophobic cavity of MDM2. To select for the anticipated modifications we employed molecular docking. Synthesized molecules were evaluated for their ability to induce apoptosis in two cancer cell lines and their derivatives with different status of p53 (colorectal HCT116 and osteosarcoma U2OS cells) by Annexin V staining. The target activity was estimated using high-content imaging system Operetta. Valine and phenylglycine ester derivatives were identified as potentially active MDM2-p53 inhibitors.

The 26S proteasome is a multifaceted target for anti-cancer therapies.

Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications.