Antibodies targeting PD-1 and PD-L1 have achieved considerable success against various cancers, however their effectiveness is limited as both therapeutic and diagnostic tools. Meanwhile, small organic molecules and macrocyclic peptides are currently at various stages of development as modulators of the PD-1/PD-L1 axis. Here, we report an array of low molecular weight anti-PD-L1 macrocyclic peptides, among which two─FM2 and FM213─emerged as promising bifunctional suppressors of PD-1/PD-L1 binding. Through a compound-centric proteomic approach, we demonstrated that FM213 robustly binds to PD-L1 in NSCLC cells and exosomes. Furthermore, at its highest non-cytotoxic concentration (2 μM), FM213 (i) significantly enhances recognition and destruction of NSCLC cells by human PBMCs, and (ii) promotes the internalization of cell-surface PD-L1 into the cytosol, leading to its degradation via a lysosome-dependent pathway. Finally, coinhibition of TIGIT and PD-L1 by DTBP-3, a TIGIT inhibitor, and FM213, respectively, enhances the antitumor immunity of anti-PD-L1 ligands.

Abstract Immune checkpoint inhibitors (ICIs) targeting the PD-1-PD-L1 axis have revolutionized cancer therapy. However, their limited clinical success and the emergence of resistance mechanisms highlight the need for novel strategies to enhance anti-PD-1-PD-L1 immunotherapy. In this context, Iridium (III) complexes (Ir(III) complexes), have gained increasing attention for their potent anticancer activity and favorable safety profile. They are shown to induce immunogenic cell death (ICD) in non-small cell lung cancer (NSCLC), boosting tumor immunogenicity and improving response to conventional therapy. Here, we investigate whether Ir(III) complexes can synergize with anti-PD-L1 therapy and consequently synthetize a small array of triazine-based PD-L1 ligand-Ir(III) complexes by conjugating our previously reported anti-PD-L1 ligand (compound 10) with a bis-phenyl-pyridine-Ir(III) complex 2. The antitumor and immunomodulatory activity of Ir(III) complex 2 and PD-L1 inhibitor compound 10, as well as of four other PD-L1 ligand-Ir(III) complex conjugates 3-6, was tested in NSCLC cell lines expressing different levels of PD-L1, as well as in co-culture assays with peripheral blood mononuclear cells (PBMCs). Ir(III) complex conjugate 3 significantly enhanced anti-PD-L1-mediated PBMC tumor cell recognition and elimination, by increasing ER stress-mediated calreticulin (CRT) exposure, mitochondrial ROS production, and immunogenic signal release (ATP, HMGB1). More importantly, these effects were abrogated in PD-L1 knockout NSCLC cells incubated with the PD-L1 ligand-Ir(III) complex conjugate 3, validating a PD-L1-mediated selective delivery and dependent mechanism. These findings provide strong evidence that Ir(III) complexes potentiate anti-PD-L1 therapy in NSCLC, supporting the clinical implementation of PD-L1 ligand-Ir(III) conjugates as a novel combinatorial immunotherapeutic strategy for enhancing anti-PD-L1 therapy. Citation Format: Valentina Pagliara, Giulia Assoni, Giovanna Polcaro, Luigi Liguori, Pierfausto Seneci, Francesco Saverio Di Leva, Vincenzo Maria D’Amore, Cristina R. Ferrone, Stefano Pepe, Luciana Marinelli, Daniela Arosio, Francesco Sabbatino. Enhancing NSCLC susceptibility to anti-PD-1 - PD-L1 therapy trough PD-L1 ligand-Ir(III) complex conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6535.

HER2 overexpression/amplification (HER2+) occurs in approximately 15-20% of breast cancer (BC) and identifies a highly aggressive BC subtype. The cure rate of HER2 + BC has been significantly increased through recent clinical achievements; however, a non-negligible proportion of patients still either fails to respond or acquires resistance to targeted therapies, highlighting the need for novel treatment strategies. As demonstrated in robust preclinical studies, HER2 + BC is considered a neoplastic disease with a peculiar lipogenic phenotype, due to its crucial addiction to an exacerbated need for fatty acids (FAs) produced via FA synthase (FASN), the central lipogenic enzyme required for intracellular de novo FA biosynthesis. FASN is overexpressed/activated in most HER2 + BC cells sustaining their growth, proliferation, and aggressiveness through a reciprocal direct interplay with the HER2-driven oncogenic signaling. Recent evidence shows that rewiring of lipid metabolism in the presence of pharmacological HER2 inhibition impairs FASN up-regulation and activates the compensatory lipid metabolic pathway of FA uptake via the altered expression/activity of the transmembrane CD36 FA transporter. Thus, the latter is emerging as a potentially new and targettable mechanism of resistance to anti-HER2 therapies. Due to the limited availability of drug-like compounds that selectively target CD36, in this study we screened a library of commercial compounds through in silico docking on the crystal structure of the CD36 extracellular domain. We evaluated their chemical-physical, biological and metabolic properties through microscale thermophoresis and molecular dynamics analyses, cell viability assays performed in monotherapy and dual blockade, and gas chromatography-flame ionization detector and BODIPY C16 uptake analyses. Among the best ranked compounds, we selected two promising hits with micromolar affinity for CD36, showing in vitro that they decrease per se the proliferation of HER2 + BC cells resistant to anti-HER2 agents, induce apoptotic effects, significantly reduce FA intracellular internalization, and potentiate the cytotoxic activity of lapatinib, i.e. the most suitable anti-HER2 drug used in in vitro bioassays. Taken together, these findings support that our novel anti-CD36 small molecules should undergo hit-to-lead optimization to prospectively improve the efficacy of anti-HER2 agents in HER2 + BC refractory to targeted therapy.

Human antigen R (HuR) is an RNA binding protein (RBP) belonging to the ELAV (Embryonic Lethal Abnormal Vision) family, which stabilizes mRNAs and regulates the expression of multiple genes. Its altered expression or localization is related to pathological features such as cancer or inflammation. Dihydrotanshinone I (DHTS I) is a naturally occurring, tetracyclic ortho-quinone inhibitor of the HuR-mRNA interaction. Our earlier efforts led to the identification of a synthetic Tanshinone Mimic (TM) 2 with improved affinity for HuR. Here we report five new TM probes 3-5 bearing a detection-promoting moiety (either photo affinity probe - PAP or biotin) as a para-substituent on the phenyl-sulphonamide for mechanism of action (MoA) studies. Biological and biochemical assays were used to characterize the novel TM conjugates 3-5. They showed similar toxic activity in HuR-expressing triple-negative breast cancer MDA-MB-231 cells, with micromolar CC50s. REMSAs revealed that photoactivatable groups (4 a and 4 b), but not biotin (5 a and 5 b), prevented conjugates' ability to disrupt rHuR-RNA complexes. Further biochemical studies confirmed that biotinylated probes, in particular 5 a, can be used to isolate rM1 M2 from solutions, taking advantage of streptavidin-coated magnetic beads, thus being the most promising HuR inhibitor to be used for further MoA studies in cell lysates.

The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can lead to beneficial anti-inflammatory effects. To this aim, we previously synthesized a novel class of small molecules named Tanshinone Mimics (TMs) able to interfere with HuR-RNA binding, and that dampen the LPS-induced immune response. Herein, we present a novel series of TMs, encompassing thiophene 3/TM9 and 4/TM10, furan 5/TM11 and 6/TM12, pyrrole 7b/TM13, and pyrazole 8. The furan-containing 5(TM11) showed the greatest inhibitory effect of the series on HuR-RNA complex formation, as suggested by RNA Electromobility Shift Assay and Time-Resolved FRET. Molecular Dynamics Calculation of HuR - 5/TM11 interaction, quantum mechanics approaches and Surface Plasmon Resonance data, all indicates that, within the novel heteroaryl substituents, the furan ring better recapitulates the chemical features of the RNA bound to HuR. Compound 5/TM11 also showed improved aqueous solubility compared to previously reported TMs. Real-time monitoring of cell growth and flow cytometry analyses showed that 5/TM11 preferentially reduced cell proliferation rather than apoptosis in murine macrophages at immunomodulatory doses. We observed its effects on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages, showing that 5/TM11 significantly reduced the expression of proinflammatory cytokines as Cxcl10 and Il1b.

Salicylaldehyde (SA) derivatives are emerging as useful fragments to obtain reversible-covalent inhibitors interacting with the lysine residues of the target protein. Here the SA installation at the C terminus of an integrin-binding cyclopeptide, leading to enhanced ligand affinity for the receptor as well as stronger biological activity in cultured glioblastoma cells is reported.

Today it is widely recognized that the PD-1/PD-L1 axis plays a fundamental role in escaping the immune system in cancers, so that anti-PD-1/PD-L1 antibodies have been evaluated for their antitumor properties in more than 1000 clinical trials. As a result, some of them have entered the market revolutionizing the treatment landscape of specific cancer types. Nonetheless, a new era based on the development of small molecules as anti PD-L1 drugs has begun. There are, however, some limitations to advancing these compounds into clinical stages including the possible difficulty in counteracting the PD-1/PD-L1 interaction in vivo, the discrepancy between the in vitro IC50 (HTFR assay) and cellular EC50 (immune checkpoint blockade co-culture assay), and the differences in ligands’ affinity between human and murine PD-L1, which can affect their preclinical evaluation. Here, an extensive theoretical study, assisted by MicroScale Thermophoresis binding assays and NMR experiments, was performed to provide an atomistic picture of the binding event of three representative biphenyl-based compounds in both human and murine PD-L1. Structural determinants of the species’ specificity were unraveled, providing unprecedented details useful for the design of next generation anti-PD-L1 molecules.

Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases.

The RNA binding protein Human Antigen R (HuR) has been identified as a main regulator of the innate immune response and its inhibition can have beneficial anti-inflammatory potential. To inhibit HuR function of binding RNA, we previously synthesised a novel class of small molecules called Tanshinone Mimics (TMs), showing promising inhibitory activity against HuR and modulating the immune response in murine macrophages. However, limited solubility posed challenges for TM-based therapies. In this study, we aimed to enhance the hydrophilicity of TMs by introducing a 5-membered heterocycles rings on the lead molecule of Tanshinone mimics. We identified the furan containing TM11 as the most soluble compound of the series but capable of blocking the HuR-RNA complex formation. Cellular assays, revealed its potential in inducing cell cycle blockage rather than apoptosis in murine macrophages. Additionally, we investigated TM11's impact on the innate immune response triggered by lipopolysaccharide (LPS) in macrophages. Our results demonstrate that TM11 significantly reduces inflammation, even in a simulated therapeutic regime, with drug administration following two hours of LPS stimulation.

Immunotherapy has emerged as a game-changing approach for cancer treatment. Although monoclonal antibodies (mAbs) targeting the programmed cell death protein 1/programmed cell death protein 1 ligand 1 (PD-1/PD-L1) axis have entered the market revolutionizing the treatment landscape of many cancer types, small molecules, although presenting several advantages including the possibility of oral administration and/or reduced costs, struggled to enter in clinical trials, suffering of water insolubility and/or inadequate potency compared with mAbs. Thus, the search for novel scaffolds for both the design of effective small molecules and possible synergistic strategies is an ongoing field of interest. In an attempt to find novel chemotypes, a virtual screening approach was employed, resulting in the identification of new chemical entities with a certain binding capability, the most versatile of which was the benzimidazole-containing compound 10. Through rational design, a small library of its derivatives was synthesized and evaluated. The homogeneous time-resolved fluorescence (HTRF) assay revealed that compound 17 shows the most potent inhibitory activity (IC50 ) in the submicromolar range and notably, differently from the major part of PD-L1 inhibitors, exhibits satisfactory water solubility properties. These findings highlight the potential of benzimidazole-based compounds as novel promising candidates for PD-L1 inhibition.

Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA binding protein that binds and regulates the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS-induced response. Here we investigated how and to what extent small molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. We show TMs exist in solution in keto-enolic tautomerism and that, by molecular dynamic calculations, the orto quinone form is the bioactive species interacting with HuR and inhibiting its binding mode vs mRNA targets. A chemical blockage of the diphenolic, reduced form as a diacetate caused the loss of activity of TMs in vitro but resulted to prodrug-like activity in vivo . The murine macrophage cell line RAW264.7 was treated with LPS and TMs, and the modulation of cellular LPS-induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes, and that TMs reduced such interactions. Functional annotation addressed a specific set of genes involved in chemotaxis and immune response, such as Cxcl10, Il1b, Cd40 , and Fas , with a decreased association with HuR, a reduction of their expression and protein secretion. The same effect was observed in primary murine bone marrow-derived macrophages, and in vivo in an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such as TM7nox with remarkable anti-inflammatory properties in vivo .

The disaccharide trehalose is a well-established autophagy inducer, but its therapeutic application is severely hampered by its low potency and poor pharmacokinetic profile. Thus, we targeted the rational design and synthesis of trehalose-based small molecules and nano objects to overcome such issues. Among several rationally designed trehalose-centered putative autophagy inducers, we coupled trehalose via suitable spacers with known self-assembly inducer squalene to yield two nanolipid-trehalose conjugates. Squalene is known for its propensity, once linked to a bioactive compound, to assemble in aqueous media in controlled conditions, internalizing its payload and forming nanoassemblies with better pharmacokinetics. We assembled squalene conjugates to produce the corresponding nanoassemblies, characterized by a hydrodynamic diameter of 188 and 184 nm and a high stability in aqueous media as demonstrated by the measured Z-potential. Moreover, the nanoassemblies were characterized for their toxicity and capability to induce autophagy in vitro.

The Human antigen R (HuR) protein is an RNA-binding protein, ubiquitously expressed in human tissues, that orchestrates target RNA maturation and processing both in the nucleus and in the cytoplasm. A survey of known modulators of the RNA-HuR interactions is followed by a description of its structure and molecular mechanism of action - RRM domains, interactions with RNA, dimerization, binding modes with naturally occurring and synthetic HuR inhibitors. Then, the review focuses on HuR as a validated molecular target in oncology and briefly describes its role in inflammation. Namely, we show ample evidence for the involvement of HuR in the hallmarks and enabling characteristics of cancer, reporting findings from in vitro and in vivo studies; and we provide abundant experimental proofs of a beneficial role for the inhibition of HuR-mRNA interactions through silencing (CRISPR, siRNA) or pharmacological inhibition (small molecule HuR inhibitors).(c) 2021 Elsevier B.V. All rights reserved.

The inhibition of the PD-1/PD-L1 axis by monoclonal antibodies has achieved remarkable success in treating a growing number of cancers. However, a novel class of small organic molecules, with BMS-202 (1) as the lead, is emerging as direct PD-L1 inhibitors. Herein, we report a series of 2,4,6-tri- and 2,4-disubstituted 1,3,5-triazines, which were synthesized and assayed for their PD-L1 binding by NMR and homogeneous time-resolved fluorescence. Among them, compound 10 demonstrated to strongly bind with the PD-L1 protein and challenged it in a co-culture of PD-L1 expressing cancer cells (PC9 and HCC827 cells) and peripheral blood mononuclear cells enhanced antitumor immune activity of the latter. Compound 10 significantly increased interferon γ release and apoptotic induction of cancer cells, with low cytotoxicity in healthy cells when compared to 1, thus paving the way for subsequent preclinical optimization and medical applications.

A small set of trehalose-centered putative autophagy inducers was rationally designed and synthesized, with the aim to identify more potent and bioavailable autophagy inducers than free trehalose, and to acquire information about their molecular mechanism of action. Several robust, high yield routes to key trehalose intermediates and small molecule prodrugs (2-5), putative probes (6-10) and inorganic nanovectors (12a-thiol-PEG-triazoletrehalose constructs 11) were successfully executed, and compounds were tested for their autophagy-inducing properties. While small molecules 2-11 showed no pro-autophagic behavior at sub-millimolar concentrations, trehalose-bearing PEG-AuNPs 12a caused measurable autophagy induction at an estimated 40 mu M trehalose concentration without any significant toxicity at the same concentration.

In recognition of the key role played by integrins in several life-threatening dysfunctions, the search for novel small-molecule probes that selectively recognize these surface receptors is still open and widely pursued. Inspired by previously established aminoproline (Amp)-RGD based cyclopeptidomimetics with attracting alpha(V)beta(3)integrin affinity and selectivity, the design and straightforward synthesis of 18 new AmpRGD chemotypes bearing additional structural variants were herein implemented, to shift toward peptide-like alpha(V)beta(6)integrin targeted binders. The ligand competence of the synthesized products toward alpha(V)beta(6)was evaluated in competitive binding assays on isolated receptors, and alpha(V)beta(6)/alpha(V)beta(3)selectivity was determined for a subgroup of compounds, resulting in the identification of four very promising candidates. SAR considerations and docking simulations allowed us to appreciate the key structural features responsible for the observed activity.

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αvβ3 and α5β1 receptors using biotinylated vitronectin (αvβ3) and fibronectin (α5β1) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αvβ3 over α5β1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvβ3). The two RGD ligands showed IC50 values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC50 value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αVβ3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.

The use of multimeric ligands is considered as a promising strategy to improve tumor targeting for diagnosis and therapy. Herein, tetrameric RGD peptidomimetics were designed to target α v β 3 integrin-expressing tumor cells. These compounds were prepared via an oxime chemoselective assembly of cyclo (DKP-RGD) ligands and a cyclodecapeptide scaffold that allows a tetrameric presentation. The resulting tetrameric RGD peptidomimetics were shown to improve α v β 3 integrin binding compared to the monomeric form. Interestingly, these compounds were also able to enhance tumor cell endocytosis in the same way as tetrameric RGD peptides. Altogether, the results show the potential of the tetrameric cyclo (DKP-RGD) ligands for in vivo imaging and drug delivery.