DGKα/ζ inhibitors combine with PD-1 checkpoint therapy to promote T cell-mediated antitumor immunity.

Programmed cell death protein 1 (PD-1) immune checkpoint blockade therapy has revolutionized cancer treatment. Although PD-1 blockade is effective in a subset of patients with cancer, many fail to respond because of either primary or acquired resistance. Thus, next-generation strategies are needed to expand the depth and breadth of clinical responses. Toward this end, we designed a human primary T cell phenotypic high-throughput screening strategy to identify small molecules with distinct and complementary mechanisms of action to PD-1 checkpoint blockade. Through these efforts, we selected and optimized a chemical series that showed robust potentiation of T cell activation and combinatorial activity with αPD-1 blockade. Target identification was facilitated by chemical proteomic profiling with a lipid-based photoaffinity probe, which displayed enhanced binding to diacylglycerol kinase α (DGKα) in the presence of the active compound, a phenomenon that correlated with the translocation of DGKα to the plasma membrane. We further found that optimized leads within this chemical series were potent and selective inhibitors of both DGKα and DGKζ, lipid kinases that constitute an intracellular T cell checkpoint that blunts T cell signaling through diacylglycerol metabolism. We show that dual DGKα/ζ inhibition amplified suboptimal T cell receptor signaling mediated by low-affinity antigen presentation and low major histocompatibility complex class I expression on tumor cells, both hallmarks of resistance to PD-1 blockade. In addition, DGKα/ζ inhibitors combined with αPD-1 therapy to elicit robust tumor regression in syngeneic mouse tumor models. Together, these findings support targeting DGKα/ζ as a next-generation T cell immune checkpoint strategy.

Discovery of MK-8318, a Potent and Selective CRTh2 Receptor Antagonist for the Treatment of Asthma.

A novel series of tricyclic tetrahydroquinolines were identified as potent and selective CRTh2 receptor antagonists. The agonism and antagonism switch was achieved through structure-based drug design (SBDD) using a CRTh2 receptor homologue model. The challenge of very low exposures in pharmacokinetic studies was overcome by exhaustive medicinal chemistry lead optimization through focused SAR studies on the tricyclic core. Further optimization resulted in the identification of the preclinical candidate 4-(cyclopropyl((3aS,9R,9aR)-7-fluoro-4-(4-(trifluoromethoxy)benzoyl)-2,3,3a,4,9,9a-hexahydro-1H-cyclopenta[b]quinolin-9-yl)amino)-4-oxobutanoic acid (15c, MK-8318) with potent and selective CRTh2 antagonist activity and a favorable PK profile suitable for once daily oral dosing for potential treatment of asthma.

The synthesis of 2,3,6-trisubstituted 1-oxo-1,2-dihydroisoquinolines as potent CRTh2 antagonists.

New synthetic methods were developed for the preparation of 2,3,6-trisubstituted 1-oxo-1,2-dihydroisoquinolines as CRTh2 antagonists. The isoquinolinone core could be constructed before the introduction of substitution groups or synthesized through a catalytic intramolecular cyclization reaction with desired substitution groups properly installed. These synthetic strategies have helped to accelerate the SAR development of this series, and potent lead compounds were identified in both the CRTh2 receptor binding assay and the CD11b biomarker assay.

Identification of highly potent and selective PI3Kδ inhibitors.

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.

The effect of cadmium on the growth and antioxidant response for freshwater algae Chlorella vulgaris.

The objective of the present work was to evaluate the effect of exogenously applied cadmium on the physiological response of green algae Chlorella vulgaris. The study investigated the long-term effect (18 days) of cadmium on the levels of algae biomass, assimilation pigment composition, soluble protein, oxidative status (production of hydrogen peroxide and superoxide anion), antioxidant enzymes (such as superoxide dismutase, peroxidase, catalase and glutathione reductase enzyme) in C. vulgaris. The results showed that growth, the amount of chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids gradually decreased with increasing cadmium over 18 days exposure. Cadmium at concentration of 7 mg L(-1) inhibited algal growth expressed as the number of cells. Our research found that C. vulgaris has a high tolerance to cadmium. Contents of chlorophylls (Chl a and Chl b) and carotenoids (Car) of C. vulgaris was significantly decline with rising concentration of cadmium (p < 0.05). The decrease of 54.04 and 93.37 % in Chl a, 60.65 and 74.32 % in Chl b, 50.00 and 71.88 % in total carotenoids was noticed following the treatment with 3 and 7 mg L(-1) cadmium doses compared with control treatment, respectively. Cadmium treatments caused a significant change in the physiological competence (calculated as chlorophyll a/b) which increased with increasing Cd(II) doses up to 1 mg L(-1) but decreased at 3 mg L(-1). While accumulation of soluble protein was enhanced by presence of cadmium, the treatment with cadmium at 3 and 7 mg L(-1) increased the concentration of soluble proteins by 88, 95.8 % in C. vulgaris, respectively. Moreover, low doses of cadmium stimulated enzymatic (superoxide dismutase, catalase and glutathione reductase) in C. vulgaris, The content of peroxidase increased with the increasing cadmium concentration, and had slightly decreased at the concentration of 7 mg L(-1), but was still higher than control group, which showed that cadmium stress at high concentration mainly peroxidase works in C. vulgaris. And therefore, suppressed reactive oxygen species (hydrogen peroxide and superoxide) accumulated. The present study also showed that cadmium increased oxidative stress and induced antioxidant defense systems against reactive oxygen species. The observation in here analyzed C. vulgaris after exposure to cadmium indicate that hydrogen peroxide, superoxide and peroxidase in the alga with exposure to Cd(II) seemed to be parameters as biomarkers for metal-induced oxidative stress.

Isolation, structure elucidation and antibacterial activity of a new tetramic acid, ascosetin.

The ever-increasing bacterial resistance to clinical antibiotics is making many drugs ineffective and creating significant treatment gaps. This can be only circumvented by the discovery of antibiotics with new mechanisms of action. We report here the identification of a new tetramic acid, ascosetin, from an Ascomycete using the Staphylococcus aureus fitness test screening method. The structure was elucidated by spectroscopic methods including 2D NMR and HRMS. Relative stereochemistry was determined by ROESY and absolute configuration was deduced by comparative CD spectroscopy. Ascosetin inhibited bacterial growth with 2-16 µg ml(-1) MIC values against Gram-positive strains including methicillin-resistant S. aureus. It also inhibited the growth of Haemophilus influenzae with a MIC value of 8 µg ml(-1). It inhibited DNA, RNA, protein and lipid synthesis with similar IC50 values, suggesting a lack of specificity; however, it produced neither bacterial membrane nor red blood cell lysis. It showed selectivity for bacterial growth inhibition compared with fungal but not mammalian cells. The isolation, structure and biological activity of ascosetin have been detailed here.

Quality by design (QbD) of amide isosteres: 5,5-Disubstituted isoxazolines as potent CRTh2 antagonists with favorable pharmacokinetic and drug-like properties.

Isoxazoles are frequently used amide isosteres, as shown in the context of discovery of CRTh2 antagonists from amide 1 to isoxazole 2. However, persistent agonism and poor solubility in isoxazole series presented challenges to its further development. Based on the concept of quality by design (QbD), 5,5-disubstituted isoxazolines 3 were introduced. The chirality at 5 position of isoxazolines controlled the switch between two modes of actions, which led to a novel series of pure antagonists. This non-planar motif also conferred a change of shape of these molecules, which avoided flat structures and improved their physical properties.

Synthesis and SAR development of novel P2X7 receptor antagonists for the treatment of pain: part 2.

Novel P2X(7) antagonists were developed using a purine scaffold. These compounds were potent and selective at the P2X(7) receptor in human and rodent as well as efficacious in rodent pain models. Compound 15a was identified to have oral potency in several pain models in rodent similar to naproxen, gabapentin and pregabalin. Structure-activity relationship (SAR) development and results of pain models are presented.

Synthesis and SAR development of novel P2X7 receptor antagonists for the treatment of pain: part 1.

Structure-activity relationship (SAR) efforts around our initial lead compound 1 led to the identification of potent P2X(7) receptor antagonists with improved pharmacokinetic profiles. These compounds were potent and selective at the P2X(7) receptor in both human and rodent. Compound (entry 31) exhibited oral efficacy in the rat MIA and CCI pain models.

Synthesis and structure-activity relationships of heteroaryl substituted-3,4-diamino-3-cyclobut-3-ene-1,2-dione CXCR2/CXCR1 receptor antagonists.

Comprehensive SAR studies were undertaken in the 3,4-diaminocyclobut-3-ene-1,2-dione class of CXCR2/CXCR1 receptor antagonists to explore the role of the heterocycle on chemokine receptor binding affinities, functional activity, as well as oral exposure in rat. The nature of the heterocycle as well as the requisite substitution pattern around the heterocycle was shown to have a dramatic effect on the overall biological profile of this class of compounds. The furyl class, particularly the 4-halo adducts, was found to possess superior binding affinities for both the CXCR2 and CXCR1 receptors, functional activity, as well as oral exposure in rat versus other heterocyclic derivatives.

3,4-Diamino-2,5-thiadiazole-1-oxides as potent CXCR2/CXCR1 antagonists.

A series of novel and potent 3,4-diamino-2,5-thiadiazole-1-oxides were prepared and found to show excellent binding affinities for CXCR2 and CXCR1 receptors and excellent inhibitory activity of Gro-alpha and IL-8 mediated in vitro hPMN MPO release of CXCR2 and CXCR1 expressing cell lines. On the other hand, a closely related 3,4-diamino-2,5-thiadiazole-dioxide did not show functional activity despite its excellent binding affinities for CXCR2 and CXCR1 in membrane binding assays. A detailed SAR has been discussed in these two closely related structures.

Pharmacological characterization of Sch527123, a potent allosteric CXCR1/CXCR2 antagonist.

In neutrophils, growth-related protein-alpha (CXCL1) and interleukin-8 (CXCL8), are potent chemoattractants (Cytokine 14:27-36, 2001; Biochemistry 42:2874-2886, 2003) and can stimulate myeloperoxidase release via activation of the G protein-coupled receptors CXCR1 and CXCR2. The role of CXCR1 and CXCR2 in the pathogenesis of inflammatory responses has encouraged the development of small molecule antagonists for these receptors. The data presented herein describe the pharmacology of 2-hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methyl-furan-2-yl)-propyl]amino]-3,4-dioxo-cyclobut-1-enylamino}-benzamide (Sch527123), a novel antagonist of both CXCR1 and CXCR2. Sch527123 inhibited chemokine binding to (and activation of) these receptors in an insurmountable manner and, as such, is categorized as an allosteric antagonist. Sch527123 inhibited neutrophil chemotaxis and myeloperoxidase release in response to CXCL1 and CXCL8 but had no effect on the response of these cells to C5a or formyl-methionyl-leucyl-phenylalanine. The pharmacological specificity of Sch527123 was confirmed by testing in a diversity profile against a panel of enzymes, channels, and receptors. To measure compound affinity, we characterized [(3)H]Sch527123 in both equilibrium and nonequilibrium binding analyses. Sch527123 binding to CXCR1 and CXCR2 was both saturable and reversible. Although Sch527123 bound to CXCR1 with good affinity (K(d) = 3.9 +/- 0.3 nM), the compound is CXCR2-selective (K(d) = 0.049 +/- 0.004 nM). Taken together, our data show that Sch527123 represents a novel, potent, and specific CXCR2 antagonist with potential therapeutic utility in a variety of inflammatory conditions.

Identification of full, partial and inverse CC chemokine receptor 3 agonists using [35S]GTPgammaS binding.

Study of the CC chemokine receptor 3 (CCR3) has been limited to using radiolabeled agonist chemokines. A small molecule CCR3 antagonist, 2-[(6-amino-2-benzothiazolyl)thio]-N-[1-[(3,4-dichlorylphenyl)methyl]-4-piperidinyl]acetamide, Banyu (I), was tritiated and used for pharmacological studies. Banyu (I) has a K(d) of 5.0+/-0.4 and 4.3+/-1.8 nM on human CCR3 transfectants and eosinophils, and noncompetitively inhibits [125I]eotaxin binding and eotaxin-induced [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. The proportion of [125I]eotaxin: [3H]Banyu (I) binding sites in eosinophils or transfectants was 35% or 13%, although both binding sites were overexpressed in transfectants. CCR3 spontaneously couples to G-proteins in CCR3 transfectants, demonstrated by changes in basal and eotaxin-induced [35S]GTPgammaS binding under reduced NaCl and GDP concentrations. Consequently, Banyu (I) was identified as an inverse agonist. In contrast, CCL18 and I-TAC (interferon-inducible T cell alpha-chemoattractant) were neutral antagonists, inhibiting eotaxin-induced [35S]GTPgammaS binding, with minimal effect on basal coupling of CCR3 to G proteins. Eotaxin, eotaxin-2 and monocyte chemoattractant protein (MCP)-4 are full agonists inducing [35S]GTPgammaS binding; eotaxin-3, MCP-3, RANTES (regulated on activation normal T cell expressed and secreted), vMIP-I (Kaposi's sarcoma-associated herpesvirus macrophage inflammatory protein-) and vMIP-II are partial agonists, indicating that this is a sensitive method to quantitate agonist efficacy.