Several combinatorial methods have been developed to create focused or diverse chemical libraries with a wide range of linear or macrocyclic chemical molecules: peptides, non-peptide oligomers, peptidomimetics, small-molecules, and natural product-like organic molecules. Each combinatorial approach has its own unique high-throughput screening and encoding strategy. In this article, we provide a brief overview of combinatorial chemistry in drug discovery with emphasis on recently developed new technologies for design, synthesis, screening and decoding of combinatorial library. Examples of successful application of combinatorial chemistry in hit discovery and lead optimization are given. The limitations and strengths of combinatorial chemistry are also briefly discussed. We are now in a better position to truly leverage the power of combinatorial technologies for the discovery and development of next-generation drugs.

Cancer is one of the major and leading causes of death worldwide. Two of the greatest challenges in fighting cancer are early detection and effective treatments with no or minimum side effects. Widespread use of targeted therapies and molecular imaging in clinics requires high affinity, tumor-specific agents as effective targeting vehicles to deliver therapeutics and imaging probes to the primary or metastatic tumor sites. Combinatorial libraries such as phage-display and one-bead one-compound (OBOC) peptide libraries are powerful approaches in discovering tumor-targeting peptides. This review gives an overview of different combinatorial library technologies that have been used for the discovery of tumor-targeting peptides. Examples of tumor-targeting peptides identified from each combinatorial library method will be discussed. Published tumor-targeting peptide ligands and their applications will also be summarized by the combinatorial library methods and their corresponding binding receptors.

Abstract Background. Currently, there are no effective medications that reverse periodontal disease (PD)-induced bone loss. The objective of this study was to test a new anabolic compound, LLP2A-Ale, or with the combination treatment of mesenchymal stromal cell (MSC), in the treatment of bone loss secondary to PD. Methods. PD was induced in mice by placing a ligature around the second right molar. At one week after disease induction, the mice were treated with placebo, LLP2A-Ale, MSCs, or combination of LLP2A-Ale + MSCs, and euthanized at week 4. Results. We found that PD induced alveolar bone loss that was associated with reduced bone formation. LLP2A alone and in combination with MSCs sustained alveolar bone formation and reversed alveolar bone loss. Additionally, PD alone caused systemic inflammation and increased the circulating levels of G-CSF, IP-10, MIP-1a, and MIP2, which were suppressed by LLP2A-Ale +/- MSCs. LLP2A-Ale +/- MSCs increased bone formation at the peripheral skeletal site (distal femur), which was otherwise suppressed by PD. Conclusion. Our findings indicated that LLP2A-Ale treatment rescued alveolar bone loss caused by PD, primarily by increasing bone formation. LLP2A-Ale also attenuated the circulating levels of a series of inflammatory cytokines and reversed the PD-induced suppression of systemic bone formation.

Background

Currently, there are no effective medications to reverse periodontal disease (PD)-induced bone loss. The objective of this study was to test a new anabolic compound, LLP2A-Ale, or with the combination treatment of mesenchymal stromal cell (MSC), in the treatment of bone loss secondary to PD.

Methods

PD was induced in mice by placing a ligature around the second right molar. At one week after disease induction, the mice were treated with placebo, LLP2A-Ale, MSCs, or combination of LLP2A-Ale + MSCs, and euthanized at week 4.

Results

We found that PD induced alveolar bone loss that was associated with reduced bone formation. LLP2A-Ale alone or in combination with MSCs sustained alveolar bone formation and reversed alveolar bone loss. Additionally, PD alone caused systemic inflammation and increased the circulating levels of G-CSF, IP-10, MIP-1a, and MIP2, which were suppressed by LLP2A-Ale +/- MSCs. LLP2A-Ale +/- MSCs increased bone formation at the peripheral skeletal site (distal femur), which was otherwise suppressed by PD.

Conclusion

Our findings indicated that LLP2A-Ale treatment rescued alveolar bone loss caused by PD, primarily by increasing bone formation. LLP2A-Ale also attenuated the circulating levels of a series of inflammatory cytokines and reversed the PD-induced suppression of systemic bone formation.

Through the one-bead two-compound (OB2C) ultra-high-throughput screening method, we discovered a new small-molecule compound LLS2 that can kill a variety of cancer cells. Pull-down assay and LC/MS-MS indicated that galectin-1 is the target protein of LLS2. Galectin-1 is known to be involved in the regulation of proliferation, apoptosis, cell cycle, and angiogenesis. Binding of LLS2 to galectin-1 decreased membrane-associated H-Ras and K-Ras and contributed to the suppression of pErk pathway. Importantly, combination of LLS2 with paclitaxel (a very important clinical chemotherapeutic agent) was found to exhibit synergistic activity against several human cancer cell lines (ovarian cancer, pancreatic cancer, and breast cancer cells) in vitro Furthermore, in vivo therapeutic study indicated that combination treatment with paclitaxel and LLS2 significantly inhibits the growth of ovarian cancer xenografts in athymic mice. Our results presented here indicate that the OB2C combinatorial technology is a highly efficient drug screening platform, and LLS2 discovered through this method can be further optimized for anticancer drug development. Mol Cancer Ther; 16(7); 1212-23. ©2017 AACR.

Through the one-bead two-compound (OB2C) ultra-high-throughput screening method, we discovered a new small-molecule compound LLS2 that can kill a variety of cancer cells. Pull-down assay and LC/MS-MS indicated that galectin-1 is the target protein of LLS2. Galectin-1 is known to be involved in the regulation of proliferation, apoptosis, cell cycle, and angiogenesis. Binding of LLS2 to galectin-1 decreased membrane-associated H-Ras and K-Ras and contributed to the suppression of pErk pathway. Importantly, combination of LLS2 with paclitaxel (a very important clinical chemotherapeutic agent) was found to exhibit synergistic activity against several human cancer cell lines (ovarian cancer, pancreatic cancer, and breast cancer cells) in vitro Furthermore, in vivo therapeutic study indicated that combination treatment with paclitaxel and LLS2 significantly inhibits the growth of ovarian cancer xenografts in athymic mice. Our results presented here indicate that the OB2C combinatorial technology is a highly efficient drug screening platform, and LLS2 discovered through this method can be further optimized for anticancer drug development. Mol Cancer Ther; 16(7); 1212-23. ©2017 AACR.

Cell-biomaterial interactions are primarily governed by cell adhesion, which arises from the binding of cellular integrins to the extracellular matrix (ECM). Integrins drive the assembly of focal contacts that serve as mechanotransducers and signaling nexuses for stem cells, for example integrin α4β1 plays pivotal roles in regulating mesenchymal stem cell (MSC) homing, adhesion, migration and differentiation. The strategy to control the integrin-mediated cell adhesion to bioinspired, ECM-mimicking materials is essential to regulate cell functions and tissue regeneration. Previously, using one-bead one-compound (OBOC) combinatorial technology, we discovered that LLP2A was a high-affinity peptidomimetic ligand (IC50 = 2 pM) against integrin α4β1. In this study, we identified that LLP2A had a strong binding to human early gestation chorionic villi-derived MSCs (CV-MSCs) via integrin α4β1. To improve CV-MSC seeding, expansion and delivery for regenerative applications, we constructed artificial scaffolds simulating the structure of the native ECM by immobilizing LLP2A onto the scaffold surface as cell adhesion sites. LLP2A modification significantly enhanced CV-MSC adhesion, spreading and viability on the polymeric scaffolds via regulating signaling pathways including phosphorylation of focal adhesion kinase (FAK), and AKT, NF-kB and Caspase 9. In addition, we also demonstrated that LLP2A had strong binding to MSCs of other sources, such as bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs). Therefore, LLP2A and its derivatives not only hold great promise for improving CV-MSC-mediated treatment of fetal diseases, but they can also be widely applied to functionalize various biological and medical materials, which are in need of MSC recruitment, enrichment and survival, for regenerative medicine applications.

Oral squamous cell carcinoma has a low five-year survival rate, which may be due to late detection and a lack of effective tumor-specific therapies. Using a high throughput drug discovery strategy termed one-bead one-compound combinatorial library, the authors identified six compounds with high binding affinity to different human oral squamous cell carcinoma cell lines but not to normal cells. Current work is under way to develop these ligands to oral squamous cell carcinoma specific imaging probes or therapeutic agents.

The "one-bead two-compound" (OB2C) combinatorial library is constructed on topologically segregated trifunctional bilayer beads such that each bead has a fixed cell-capturing ligand and a random library compound co-displayed on its surface and a chemical coding tag (bar code) inside the bead. An OB2C library containing thousands to millions of compounds can be synthesized and screened concurrently within a short period of time. When live cells are incubated with such OB2C libraries, every bead will be coated with a monolayer of cells. The cell membranes of the captured cells facing the bead surface are exposed to the library compounds tethered to each bead. A specific biochemical or cellular response can be detected with an appropriate reporter system. The OB2C method enables investigators to rapidly discover synthetic molecules that not only interact with cell-surface receptors but can also stimulate or inhibit downstream cell signaling. To demonstrate this powerful method, one OB2C peptide library and two OB2C peptidomimetic libraries were synthesized and screened against Molt-4 lymphoma cells to discover "death ligands." Apoptosis of the bead-bound cells was detected with immunocytochemistry using horseradish peroxidase (HRP)-conjugated anti-cleaved caspase-3 antibody and 3,3'-diaminobenzidine as a substrate. Two novel synthetic "death ligands" against Molt-4 cells were discovered using this OB2C library approach.

The nuclear receptor RORγ is a major driver of autoimmune diseases and certain types of cancer due to its aberrant function in T helper 17 (Th17) cell differentiation and tumor cholesterol metabolism, respectively. Compound screening using the classic receptor-coactivator interaction perturbation scheme led to identification of many small-molecule modulators of RORγ(t). We report here that inverse agonists/antagonists of RORγ such as VTP-43742 derivative VTP-23 and TAK828F, which can potently inhibit the inflammatory gene program in Th17 cells, unexpectedly lack high potency in inhibiting the growth of TNBC tumor cells. In contrast, antagonists such as XY018 and GSK805 that strongly suppress tumor cell growth and survival display only modest activities in reducing Th17-related cytokine expression. Unexpectedly, we found that VTP-23 significantly induces the cholesterol biosynthesis program in TNBC cells. Our further mechanistic analyses revealed that VTP-23 enhances the local chromatin accessibility, H3K27ac mark and the cholesterol master regulator SREBP2 recruitment at the RORγ binding sites, whereas XY018 exerts the opposite activities. Yet, they display similar inhibitory effects on circadian rhythm program. Similar distinctions and contrasting activities between TAK828F and SR2211 in their effects on local chromatin structure at Il17 genes were also observed. Together, our study shows for the first-time that structurally distinct RORγ antagonists possess different or even contrasting activities in tissue/cell-specific manner. Our findings also highlight that the activities at natural chromatin are key determinants of RORγ modulators' tissue selectivity.