Structure-Based Rational Design of Constrained Peptides as TIM-3 Inhibitors.

Blocking the immunosuppressive function of T-cell immunoglobulin mucin-3 (TIM-3) is an established therapeutic strategy to maximize the efficacy of immune checkpoint inhibitors for cancer immunotherapy. Currently, effective inhibition of TIM-3 interactions relies on monoclonal antibodies (mAbs), which come with drawbacks such as immunogenicity risk, limited tumor penetration, and high manufacturing costs. Guided by the X-ray cocrystal structures of TIM-3 with mAbs, we report an in silico structure-based rational design of constrained peptides as potent TIM-3 inhibitors. The top cyclic peptide from our study (P2) binds TIM-3 with a K D value of 166.3 ± 12.1 nM as determined by surface plasmon resonance (SPR) screening. Remarkably, P2 efficiently inhibits key TIM-3 interactions with natural TIM-3 ligands at submicromolar concentrations in a panel of cell-free and cell-based assays. The capacity of P2 to reverse immunosuppression in T-cell/cancer cell cocultures, coupled with favorable in vitro pharmacokinetic properties, highlights the potential of P2 for further evaluation in preclinical models of immuno-oncology.

Lithocholic acid derivatives as potent modulators of the nuclear receptor RORγt.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor found in various tissues that plays a crucial role in the differentiation and proliferation of T helper 17 (Th17) cells, as well as in their generation of the pro-inflammatory cytokine IL-17A. RORγt represents a promising therapeutic target for autoimmune diseases, metabolic disorders, and multiple tumors. Despite extensive research efforts focused on the development of small molecule RORγt modulators, no drug candidates have advanced to phase 3 clinical trials owing to a lack of efficacy or safety margin. This outcome highlights the unmet need to optimize small molecule drug candidates targeting RORγt to develop effective therapies for autoimmune and inflammatory diseases. In this study, we synthesized and evaluated 3-oxo-lithocholic acid amidates as a new class of RORγt modulators. Our evaluation entailed biophysical screening, cellular screening in different platforms, molecular docking, and in vitro pharmacokinetic profiling. The top compound from our study (3-oxo-lithocholic acid amidate, A2) binds to RORγt at an equilibrium dissociation constant (KD) of 16.5 ± 1.34 nM based on microscale thermophoresis (MST). Assessment of the efficacy of A2 in the cellular RORγt reporter luciferase assay revealed a half-maximal inhibitory concentration (IC50) value of 225 ± 10.4 nM. Unlike 3-oxo-lithocholic acid, A2 demonstrated the ability to reduce the IL-17A mRNA expression levels in EL4 cells with RORγt expression using quantitative reverse transcriptase PCR (RT-PCR). Validation of the desirable physicochemical properties and stability of A2 sets the stage for the preclinical evaluation of this new class of RORγt modulators in animal models of autoimmune diseases.

Small Molecule Immunomodulators as Next-Generation Therapeutics for Glioblastoma.

Glioblastoma (GBM), the most aggressive astrocytic glioma, remains a therapeutic challenge despite multimodal approaches. Immunotherapy holds promise, but its efficacy is hindered by the highly immunosuppressive GBM microenvironment. This review underscores the urgent need to comprehend the intricate interactions between glioma and immune cells, shaping the immunosuppressive tumor microenvironment (TME) in GBM. Immunotherapeutic advancements have shown limited success, prompting exploration of immunomodulatory approaches targeting tumor-associated macrophages (TAMs) and microglia, constituting a substantial portion of the GBM TME. Converting protumor M2-like TAMs to antitumor M1-like phenotypes emerges as a potential therapeutic strategy for GBM. The blood-brain barrier (BBB) poses an additional challenge to successful immunotherapy, restricting drug delivery to GBM TME. Research efforts to enhance BBB permeability have mainly focused on small molecules, which can traverse the BBB more effectively than biologics. Despite over 200 clinical trials for GBM, studies on small molecule immunomodulators within the GBM TME are scarce. Developing small molecules with optimal brain penetration and selectivity against immunomodulatory pathways presents a promising avenue for combination therapies in GBM. This comprehensive review discusses various immunomodulatory pathways in GBM progression with a focus on immune checkpoints and TAM-related targets. The exploration of such molecules, with the capacity to selectively target key immunomodulatory pathways and penetrate the BBB, holds the key to unlocking new combination therapy approaches for GBM.

Design of cyclic peptides as novel inhibitors of ICOS/ICOSL interaction.

Compared to small molecules and antibodies, cyclic peptides exhibit unique biochemical and therapeutic attributes in the realm of pharmaceutical applications. The interaction between the inducible costimulator (ICOS) and its ligand (ICOSL) plays a key role in T-cell differentiation and activation. ICOS/ICOSL inhibition results in a reduction in the promotion of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. Herein, we implement the computational cPEPmatch approach to design the first examples of cyclic peptides that inhibit ICOS/ICOSL interaction. The top cyclic peptide from our approach possessed an IC50 value of 1.87 ± 0.15 µM as an ICOS/ICOSL inhibitor and exhibited excellent in vitro pharmacokinetic properties as a drug candidate. Our work will lay the groundwork for future endeavors in cancer drug discovery, with the goal of developing cyclic peptides that target the ICOS/ICOSL interaction.

Discovery of ICOS-Targeted Small Molecules Using Pharmacophore-Based Screening.

There are currently no small molecules clinically approved as immune checkpoint modulators. Besides possessing oral bioavailability, cell-penetrating capabilities and enhanced tumor penetration compared to monoclonal antibodies (mAbs), small molecules are amenable to pharmacokinetic optimization, which allows adopting flexible dosage regimens that may avoid immune-related adverse events associated with mAbs. The interaction of inducible co-stimulator (ICOS) with its ligand (ICOS-L) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. This study represents the development and validation of a virtual screening strategy to identify small molecules that bind a novel druggable binding pocket in human ICOS. We used a lipophilic canyon in the apo-structure of ICOS and the ICOS/ICOS-L interface individually as templates for molecular dynamics simulation to generate 3D pharmacophores subsequently used for virtual screening campaigns. Our strategy was successful finding a first-in-class small molecule ICOS binder (5P, KD value=108.08±26.76 µM) and validating biophysical screening platforms for ICOS-targeted small molecules. We anticipate that future structural optimization of 5P will result in the discovery of high affinity chemical ligands for ICOS.

First-in-class small molecule inhibitors of ICOS/ICOSL interaction as a novel class of immunomodulators.

The interaction of the inducible co-stimulator (ICOS) with its ligand (ICOSL) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. The ICOS/ICOSL pathway is a validated target for T-cell lymphomas induced by the proliferation of T-follicular helper (Tfh) cells. Moreover, the inhibition of ICOS/ICOSL interaction can decrease the enhancement of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. However, targeting ICOS/ICOSL interaction is currently restricted to monoclonal antibodies (mAbs) and there are no small molecules in existence that can block ICOS/ICOSL. To fill this gap, we report herein the first time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit ICOS/ICOSL interaction. Implementation of the developed TR-FRET assay in high-throughput screening (HTS) of a focused chemical library resulted in the identification of AG-120 as a first-in-class inhibitor of ICOS/ICOSL interaction. We further employed docking studies and molecular dynamics (MD) simulations to identify the plausible mechanism of blocking ICOS/ICOSL complex formation by AG-120. Using the structure-activity relationship (SAR) by catalog approach, we identified AG-120-X with an IC50 value of 4.68 ± 0.47 µM in the ICOS/ICOSL TR-FRET assay. Remarkably, AG-120-X revealed a dose-dependent ability to block ICOS/ICOSL interaction in a bioluminescent cellular assay based on co-culturing Jurkat T cells expressing ICOS and CHO-K1 cells expressing ICOSL. This work will pave the way for future drug discovery efforts aiming at the development of small molecule inhibitors of ICOS/ICOSL interaction as potential therapeutics for cancer as well as other diseases.

Ciprofloxacin Derivative-Loaded Nanoparticles Synergize with Paclitaxel Against Type II Human Endometrial Cancer.

Combinations of chemotherapeutic agents comprise a clinically feasible approach to combat cancers that possess resistance to treatment. Type II endometrial cancer is typically associated with poor outcomes and the emergence of chemoresistance. To overcome this challenge, a combination therapy is developed comprising a novel ciprofloxacin derivative-loaded PEGylated polymeric nanoparticles (CIP2b-NPs) and paclitaxel (PTX) against human type-II endometrial cancer (Hec50co with loss of function p53). Cytotoxicity studies reveal strong synergy between CIP2b and PTX against Hec50co, and this is associated with a significant reduction in the IC50 of PTX and increased G2/M arrest. Upon formulation of CIP2b into PEGylated polymeric nanoparticles, tumor accumulation of CIP2b is significantly improved compared to its soluble counterpart; thus, enhancing the overall antitumor activity of CIP2b when co-administered with PTX. In addition, the co-delivery of CIP2b-NPs with paclitaxel results in a significant reduction in tumor progression. Histological examination of vital organs and blood chemistry was normal, confirming the absence of any apparent off-target toxicity. Thus, in a mouse model of human endometrial cancer, the combination of CIP2b-NPs and PTX exhibits superior therapeutic activity in targeting human type-II endometrial cancer.

Discovery of Small-Molecule TIM-3 Inhibitors for Acute Myeloid Leukemia Using Pharmacophore-Based Virtual Screening.

T-cell immunoglobulin and mucin domain 3 (TIM-3) is a negative immune checkpoint that represents a promising target for cancer immunotherapy. Although encouraging results have been observed for TIM-3 inhibition in the context of acute myeloid leukemia (AML), targeting TIM-3 is currently restricted to monoclonal antibodies (mAbs). To fill this gap, we implemented a pharmacophore-based screening approach to identify small-molecule TIM-3 inhibitors. Our approach resulted in the identification of hit compounds with TIM-3 binding affinity. Subsequently, we used the structure-activity relationship (SAR) by a catalog approach to identify compound A-41 with submicromolar TIM-3 binding affinity. Remarkably, A-41 demonstrated the ability to block TIM-3 interactions with key ligands and inhibited the immunosuppressive function of TIM-3 using an in vitro coculture assay. This work will pave the way for future drug discovery efforts aiming at the development of small-molecule inhibitors TIM-3 for AML.

Discovery of First-in-Class Small Molecule Inhibitors of Lymphocyte Activation Gene 3 (LAG-3).

Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint that plays a key role in downregulating the immune response to cancer. Inhibition of LAG-3 interactions allows T cells to regain cytotoxic activity and reduce the immunosuppressive function of regulating T cells. We utilized a combination approach of focused screening and "SAR by catalog" to identify small molecules that function as dual inhibitors of the interactions of LAG-3 with major histocompatibility complex (MHC) class II and fibrinogen-like protein 1 (FGL1). Our top hit compound inhibited both LAG-3/MHCII and LAG-3/FGL1 interactions in biochemical binding assays with IC50 values of 4.21 ± 0.84 and 6.52 ± 0.47 µM, respectively. Moreover, we have demonstrated the ability of our top hit compound to block LAG-3 interactions in cell-based assays. This work will pave the way for future drug discovery efforts aiming at the development of LAG-3-based small molecules for cancer immunotherapy.

Development of a high-throughput TR-FRET screening assay for LAG-3/FGL1 interaction.

Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions. Fibrinogen-like protein 1 (FGL1) is a major LAG-3 functional ligand that is upregulated in various human cancers. LAG-3 positive T cells bind FGL1 expressed by cancer cells, which inhibits T-cell activation and cytokine secretion via indirect blocking of T cell receptor (TCR) signaling. High expression of LAG-3 and FGL1 in patients with solid tumors is associated with drug resistance and decreased survival in response to FDA-approved immune checkpoint inhibitors. Therefore, targeting the LAG-3/FGL1 pathway represents a promising therapeutic strategy to maximize the number of patients benefiting from checkpoint blockade therapy. However, there are no small molecules in existence that target LAG-3/FGL1 interaction. Herein, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit LAG-3/FGL1 interaction. We further demonstrate the implementation of the developed assay in screening chemical libraries of small molecules from the NCI Diversity Set VII, FDA-approved drugs, and a focused library of NF-κB modulators. This work will pave the way for drug discovery efforts focused on therapeutic targeting of LAG-3/FGL1 interaction using small molecules.

Multicomponent Petasis reaction for the identification of pyrazine based multi-target directed anti-Alzheimer's agents: In-silico design, synthesis, and characterization.

Multi-target directed ligands (MTDLs) have recently attracted significant interest due to their exceptional effectiveness against multi-factorial Alzheimer's disease. The present work described the development of pyrazine-based MTDLs using multicomponent Petasis reaction for the dual inhibition of tau-aggregation and human acetylcholinesterase (hAChE). The molecular structure of synthesized ligands was validated by 1H & 13C NMR and mass spectrometry. The screened compounds were shown to have a strong inhibitory effect at 10 µM concentration against tau-oligomerization and hAChE, but only moderate inhibitory activity against Aß42. Among all the compounds, the half-maximal inhibitory concentration (IC50) for 21 and 24 against hAChE were 0.71 µM and 1.09 µM, respectively, while they displayed half-maximal effective concentrations (EC50) values of 2.21 µM and 2.71 µM for cellular tau-oligomerization, respectively. Additionally, an MTT experiment using tau-expressing SH-SY5Y neuroblastoma cells revealed that 21 was more neuroprotective than the FDA-approved medication donepezil. Furthermore, an MD simulation study was performed to investigate the dynamics and stability of AChE-21 and AChE-24 complexes in an aqueous environment. The MM-PBSA calculations were performed to evaluate the binding of 21 and 24 with AChE, and the relative binding energy was calculated as -870.578 and -875.697 kJ mol-1, respectively. As a result, the study offered insight into the design of new MTDLs and highlighted 21 as a potential roadblock to the development of anti-AD medications.

Gut Microbial-Derived Metabolites as Immune Modulators of T Helper 17 and Regulatory T Cells.

The gut microbiota and its derived metabolites greatly impact the host immune system, both innate and adaptive responses. Gut dysbiosis and altered levels of microbiota-derived metabolites have been described in several immune-related and immune-mediated diseases such as intestinal bowel disease, multiple sclerosis, or colorectal cancer. Gut microbial-derived metabolites are synthesized from dietary compounds ingested by the host or host-produced metabolites, and additionally, some bacterial products can be synthesized de novo. In this review, we focus on the two first metabolites families including short-chain fatty acids, indole metabolites, polyamines, choline-derived compounds, and secondary bile acids. They all have been described as immunoregulatory molecules that specifically affect the adaptive immune system and T helper 17 and regulatory T cells. We discuss the mechanisms of action and the consequences in health and diseases related to these gut microbial-derived metabolites. Finally, we propose that the exogenous administration of these molecules or other compounds that bind to their immunoregulatory receptors in a homologous manner could be considered therapeutic approaches.

Thiophene derivative-loaded nanoparticles mediate anticancer activity through the inhibition of kinases and microtubule assembly.

Different tetrahydrobenzo[b]thiophene derivatives were explored as new tubulin polymerization destabilizers to arrest tumor cell mitosis. A series of compounds incorporating the tetrahydrobenzo[b]thiophene scaffold were synthesized, and their biological activities were investigated. The cytotoxicity of each of the synthesized compounds was assessed against a range of cell lines. Specifically, the benzyl urea tetrahydrobenzo[b]thiophene derivative, 1-benzyl-3-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)urea (BU17), was identified as the most potent compound with broad-spectrum antitumor activity against several cancer cell lines. The potential mechanism(s) of action were investigated where dose-dependent G2/M accumulation and A549 cell cycle arrest were detected. Additionally, A549 cells treated with BU17 expressed enhanced levels of caspase 3 and 9, indicating the induction of apoptosis. Furthermore, it was found that BU17 inhibits WEE1 kinase and targets tubulin by blocking its polymerization. BU17 was also formulated into PLGA nanoparticles, and it was demonstrated that BU17-loaded nanoparticles could significantly enhance antitumor activity compared to the soluble counterpart.

Cyclohepta[b]thiophenes as Potential Antiproliferative Agents: Design, Synthesis, In Vitro, and In Vivo Anticancer Evaluation.

Several thiophene featuring compounds are known for their promising antiproliferative activity. Prompted by the urgent need to identify new potent anticancer agents, 16 compounds of benzamides, benzylamines, and urea analogues incorporating a cyclohepta[b]thiophene scaffold were synthesized and biologically evaluated with a cell proliferation assay using the A549 nonsmall cell lung cancer cell line. Compound 17 demonstrated both potent and broad-spectrum anticancer activity with submicromolar 50% growth inhibition (GI50) values. It also showed superior antiproliferative activity (vs nocodazole) in OVACAR-4, OVACAR-5, CAKI-1, and T47D cell lines with GI50 values of 2.01 (vs 22.28), 2.27 (vs 20.75), 0.69 (vs 1.11), and 0.362 (vs 81.283) µM, respectively. Additionally, compound 17 displayed minimal cytotoxicity based on 50% lethal concentration (LC50) values toward all tested cell lines. Further cell-based mechanistic studies of compound 17 revealed its ability to induce cell cycle arrest of A549 cells as evidenced by dose dependent G2/M accumulation. Furthermore, induction of early apoptosis along with activation of caspase 3, 8, and 9 were confirmed in A549 cells treated with compound 17. Targeting tubulin polymerization may explain the mechanism of the antiproliferative activity of compound 17 based on cell cycle analysis, detected apoptosis, and in vitro inhibition of tubulin polymerization. In vitro data were further supported by in vivo antitumor efficacy studies of compound 17 in a CT26 murine model for which the results showed a reduction in the tumor growth compared to untreated mice. Overall, compound 17 has the potential to function as a promising candidate for further development of potent anticancer chemotherapeutics.

Synthesis, antimicrobial, antiquorum-sensing, antitumor and cytotoxic activities of new series of cyclopenta(hepta)[b]thiophene and fused cyclohepta[b]thiophene analogs.

New series of cyclopenta(hepta)[b]thiophene and fused cyclohepta[b]thiophene analogs were synthesized. The new analogs were assessed for antibacterial efficacy toward Escherichia coli ATCC 12435, Bacillus cereus UW 85 and Staphylococcus aureus. Compounds 5a, 6b and 12 showed eminent activity toward all selected bacterial strains compared to ampicillin. The antifungal efficacy of the same analogs was also examined toward Candida albicans and Aspergillus fumigatus 293, whereas 5a,b and 12 showed excellent efficacy toward both of the tested fungi. Moreover, 4b, 6a, 14a and 17 demonstrated interesting antifungal efficacy toward A. fumigatus. The same analogs were assessed for antiquorum-sensing efficacy toward Chromobacterium violacium ATCC 12472, whereas 5a, 12 and 15a demonstrated moderate activity. The new analogs were also esteemed for in vitro antitumor activity over HepG2, MCF-7 and HT-29 cancer cell lines. Results indicated that 6b and 10 are the most potent analogs against the three tested cell lines. In addition, 5a, 6a, 7 and 15a displayed interesting activity toward all tested cell lines. The active in vitro antitumor analogs were screened for in vivo antitumor activity over EAC in mice as well as in vitro cytotoxicity toward W138 human normal cell line. Results demonstrated that 6a,b and 10 have the highest in vivo activity, and that all tested compounds were found to be less cytotoxic than 5-FU toward W138 normal cell line. The DNA-binding affinity of the active antimicrobial and/or antitumor analogs was also assessed, whereas 4a, 5b, 10 and 15a exhibited the highest affinity. In silico studies affirmed that the inspected compounds are compatible with Lipinski's rule of five with expected good oral absorption.