Comprehensive in vitro characterization of PD-L1 small molecule inhibitors.

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has emerged as a powerful strategy in cancer immunotherapy. Recently, there have been enormous efforts to develop potent PD-1/PD-L1 inhibitors. In particular, Bristol-Myers Squibb (BMS) and Aurigene Discovery Technologies have individually disclosed several promising PD-1/PD-L1 inhibitors, whose detailed experimental data are not publicly disclosed. In this work, we report the rigorous and systematic in vitro characterization of a selected set of potent PD-1/PD-L1 macrocyclic peptide (BMSpep-57) and small-molecule inhibitors (BMS-103, BMS-142) from BMS and a peptidomimetic small-molecule inhibitor from Aurigene (Aurigene-1) using a series of biochemical and cell-based assays. Our results confirm that BMS-103 and BMS-142 are strongly active in biochemical assays; however, their acute cytotoxicity greatly compromised their immunological activity. On the other hand, Aurigene-1 did not show any activity in both biochemical and immunological assays. Furthermore, we also report the discovery of a small-molecule immune modulator, whose mode-of-action is not clear; however, it exhibits favorable drug-like properties and strong immunological activity. We hope that the results presented here will be useful in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors.

Isoniazid induces a monocytic-like phenotype in HL-60 cells.

Isoniazid (INH) is one of the oldest drugs for the treatment of tuberculosis (TB) and is of continual clinical and research interest. The aim of the current study is to investigate the ability of INH to induce monocyte differentiation and the underlying signaling pathway involved in this phenomenon using HL-60 cells. In this study, HL-60 cells were treated with different non-cytotoxic concentrations of INH or vitamin D (a well-known inducer of monocytic differentiation) to determine key functional changes in the phenotype of these cells using several biochemical and cytobiological experiments. HL-60 cells are derived from human promyelocytic leukemia and bear some resemblance to promyelocytes, which differentiate into various cell types. INH-induced differentiation was confirmed to occur in a concentration-dependent manner through several functional markers such as nonspecific esterase activity, NADPH oxidase activity and expression of surface markers CD14 and CD16 (characteristic of monocytes). INH-induced monocytic-like differentiation in HL-60 cells and demonstrated that at least 25% of cells were differentiated within the range of the pharmacological concentrations of INH. To determine the effects of INH on HL-60 cells, we applied quantitative proteomics that revealed 32 proteins were altered significantly in pathways that could involve differentiation signals. Lastly, INH activated the ERK-1/MAPK signaling pathway based on detection of phosphorylated ERK-1. These in vitro findings in HL-60 cells warrant further study using promyelocytes or hematopoietic stem cells to evaluate the physiological capability of INH to induce monocytic differentiation that may aid in host defense against TB.

Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.

Granule protein processing and regulated secretion in neutrophils.

Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines, and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First, we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking, and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.

An essential role for Rab27a GTPase in eosinophil exocytosis.

Eosinophil degranulation has been implicated in inflammatory processes associated with allergic asthma. Rab27a, a Rab-related GTPase, is a regulatory intracellular signaling molecule expressed in human eosinophils. We postulated that Rab27a regulates eosinophil degranulation. We investigated the role of Rab27a in eosinophil degranulation within the context of airway inflammation. Rab27a expression and localization in eosinophils were investigated by using subcellular fractionation combined with Western blot analysis, and the results were confirmed by immunofluorescence analysis of Rab27a and the granule membrane marker CD63. To determine the function of eosinophil Rab27a, we used Ashen mice, a strain of Rab27a-deficient animals. Ashen eosinophils were tested for degranulation in response to PAF and calcium ionophore by measuring released EPX activity. Airway EPX release was also determined by intratracheal injection of eosinophils into mice lacking EPX. Rab27a immunoreactivity colocalized with eosinophil crystalloid granules, as determined by subcellular fractionation and immunofluorescence analysis. PAF induced eosinophil degranulation in correlation with redistribution of Rab27a(+) structures, some of which colocalized with CD63(+) crystalloid granules at the cell membrane. Eosinophils from mice had significantly reduced EPX release compared with normal WT eosinophils, both in vitro and in vivo. In mouse models, Ashen mice demonstrated reduced EPX release in BAL fluid. These findings suggest that Rab27a has a key role in eosinophil degranulation. Furthermore, these findings have implications for Rab27a-dependent eosinophil degranulation in airway inflammation.

Identification and modeling of a drug target for Clostridium perfringens SM101.

In the present study, comparative genome analysis between Clostridium perfringens and the human genome was carried out to identify genes that are essential for the pathogen's survival, and non-homologous to the genes of human host, that can be used as potential drug targets. The study resulted in the identification of 426 such genes. The number of these potential drug targets thus identified is significantly lower than the genome's protein coding capacity (2558 protein coding genes). The 426 genes of C. perfringens were further analyzed for overall similarities with the essential genes of 14 different bacterial species present in Database of Essential Genes (DEG). Our results show that there are only 5 essential genes of C. perfringens that exhibit similarity with 12 species of the 14 different bacterial species present in DEG database. Of these, 1 gene was similar in 12 species and 4 genes were similar in 11 species. Thus, the study opens a new avenue for the development of potential drugs against the highly pathogenic bacterium. Further, by selecting these essential genes of C. perfringens, which are common and essential for other pathogenic microbial species, a broad spectrum anti-microbial drug can be developed. As a case study, we have built a homology model of one of the potential drug targets, ABC transporter-ATP binding protein, which can be employed for in silico docking studies by suitable inhibitors.