This study describes the rational design, synthesis, and preclinical evaluation of a new class of D(+)-biotin–conjugated resorcinol dibenzyl ether small molecules intended for tumor-targeted PD-L1 inhibition. The lead molecule, SW-1, disrupted PD-1/PD-L1 interaction with an IC50 of 5.6 nM, showed high binding affinity to both human and murine PD-L1, and exhibited favorable pharmacokinetic properties in rats. In a B16-F10 melanoma mouse model, SW-1 achieved tumor growth inhibition (TGI = 64.9%) compared with a PD-L1 antibody (TGI = 44.2%), increased CD8+ T-cell tumor infiltration, and did not produce obvious body weight loss. The authors propose SW-1 as a promising lead for precision tumor-targeted immunotherapy.
Targeting the PD-1/PD-L1 checkpoint remains central to contemporary cancer immunotherapy, but monoclonal antibodies carry limitations including prolonged systemic exposure, limited objective responses in unselected cohorts, and risk of immune-related adverse events such as checkpoint inhibitor-associated myocarditis. Small-molecule inhibitors offer advantages including oral bioavailability and tunable pharmacokinetics, potentially reducing Fc-mediated off-target effects.
A core translational challenge is the constitutive expression of PD-L1 on many normal tissues. Systemic blockade can disrupt peripheral immune tolerance and provoke irAEs. The authors therefore sought to develop PD-L1 inhibitors capable of preferential tumor accumulation to preserve PD-L1 function in healthy tissues while inhibiting intratumoral immunosuppression.
The design integrates a previously validated resorcinol dibenzyl ether PD-L1 inhibitory scaffold with a D(+)-biotin tumor-homing motif. The strategy exploits overexpression of the sodium-dependent multivitamin transporter (SMVT) in multiple tumor types (including B16-F10 melanoma), using biotin as a high-affinity endogenous ligand for receptor-mediated uptake. The authors note that biotin may also possess intrinsic immunomodulatory effects supporting dendritic cell maturation and CD8+ T-cell infiltration, potentially synergizing with PD-L1 blockade.
A series of D(+)-biotin–conjugated resorcinol dibenzyl ether derivatives (SW-1 through SW-7) were synthesized using standard organic methods with reagents from commercial suppliers. Purity of reported compounds was confirmed by HPLC and was ≥95% (area %) for all reported compounds; detailed 1H and 13C NMR, HRMS, and retention times are provided for each compound in the source. BMS-202 was used as a positive control in relevant assays.
AutoDock Vina docking and 100 ns molecular dynamics (MD) simulations (GROMACS 2019, CHARMM36, TIP3P water) were conducted for protein–ligand complexes. The production MD runs used a 2 fs timestep and standard PME electrostatics. Trajectory analyses included RMSD, RMSF, radius of gyration (Rg), SASA, hydrogen-bond counts, interaction energy, principal component analysis, and MM/PBSA estimation of binding free energy. These computational studies supported stable binding of SW-1 at the PD-L1 dimer interface with key intermolecular interactions consistent with high affinity.
An HTRF-based PD-1/PD-L1 binding assay used His-tagged PD-L1 and GST-tagged PD-1 with fluorescent reporters to quantify inhibition by serially diluted compounds in 384-well plates. Antiproliferative activity was assessed using the CCK8 assay. Across the panel, SW-1 emerged as the most potent inhibitor, with reported IC50 = 5.6 nM for disruption of PD-1/PD-L1 interaction.
SPR imaging (PlexArray HT) was used to determine binding kinetics and affinities. Recombinant human and murine PD-L1 proteins were employed. SW-1 showed high binding affinity to both species’ PD-L1 proteins, supporting translational relevance for syngeneic mouse models and cross-species target engagement.
Primary pharmacokinetic studies of SW-1 were performed in Sprague–Dawley rats (n = 6). The authors report favorable systemic exposure and a suitable half-life for in vivo application; detailed PK parameters are provided in the source article’s Table 2. These PK characteristics informed dose selection for efficacy studies.
In the B16-F10 syngeneic melanoma model, SW-1 produced a tumor growth inhibition of 64.9%, exceeding the comparator PD-L1 antibody (TGI = 44.2%). Treatment with SW-1 led to increased CD8+ T-cell infiltration into the tumor microenvironment, consistent with effective checkpoint inhibition and local immune activation. Body weight remained stable during treatment, indicating tolerability in the tested regimen.
In vivo safety assessments included monitoring of body weight and measurement of serum cardiac biomarkers; the authors report favorable safety without obvious weight loss. Serum biomarker data are summarized in the source Table 3. No overt toxicities were reported in the described preclinical evaluations.
The authors conclude that SW-1 is a highly potent biotin-conjugated small-molecule PD-L1 inhibitor with strong target activity, favorable pharmacokinetic behavior, and meaningful antitumor efficacy in a syngeneic melanoma model, combined with acceptable preclinical safety. The tumor-directed design—leveraging SMVT overexpression and biotin-mediated uptake—aims to reduce systemic PD-L1 engagement and associated irAE risk while concentrating activity in the tumor. SW-1 is proposed as a promising lead for further development in precision tumor-targeted immunotherapy.
Key methodological elements include standard organic synthesis and purification techniques, spectroscopic characterization (1H/13C NMR, HRMS), MD simulation protocols (GROMACS, CHARMM36, 100 ns production runs), an HTRF-based PD-1/PD-L1 binding assay, SPR imaging for affinity measurements, CCK8 antiproliferative assays, and in vivo pharmacokinetic and efficacy studies in rats and B16-F10 tumor-bearing mice. Where detailed numerical results and experimental parameters are reported (e.g., PK metrics, serum biomarker values), these are provided in the tables and figures of the source article.