Bogdan Musielak

Structure of the Complex of Human Programmed Death 1, PD-1, and Its Ligand PD-L1.

Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has recently provided breakthrough progress in the treatment of melanoma, non-small cell lung cancer, and other types of cancer. Small-molecule drugs interfering with this pathway are highly awaited, but their development is hindered by insufficient structural information. This study reveals the molecular details of the human PD-1/PD-L1 interaction based on an X-ray structure of the complex. First, it is shown that the ligand binding to human PD-1 is associated with significant plasticity within the receptor. Second, a detailed molecular map of the interaction surface is provided, allowing definition of the regions within both interacting partners that may likely be targeted by small molecules.

Structural basis for small molecule targeting of the programmed death ligand 1 (PD-L1).

Targeting the PD-1/PD-L1 immunologic checkpoint with monoclonal antibodies has provided unprecedented results in cancer treatment in the recent years. Development of chemical inhibitors for this pathway lags the antibody development because of insufficient structural information. The first nonpeptidic chemical inhibitors that target the PD-1/PD-L1 interaction have only been recently disclosed by Bristol-Myers Squibb. Here, we show that these small-molecule compounds bind directly to PD-L1 and that they potently block PD-1 binding. Structural studies reveal a dimeric protein complex with a single small molecule which stabilizes the dimer thus occluding the PD-1 interaction surface of PD-L1s. The small-molecule interaction “hot spots” on PD-L1 surfaces suggest approaches for the PD-1/PD-L1 antagonist drug discovery.

Small-molecule inhibitors of PD-1/PD-L1 immune checkpoint alleviate the PD-L1-induced exhaustion of T-cells.

Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition.

Two‐Step Synthesis of Complex Artificial Macrocyclic Compounds

The design and synthesis of head-to-tail linked artificial macrocycles using the Ugi-reaction has been developed. This synthetic approach of just two steps is unprecedented, short, efficient and works over a wide range of medium (8-11) and macrocyclic (≥12) loop sizes. The substrate scope and functional group tolerance is exceptional. Using this approach, we have synthesized 39 novel macrocycles by two or even one single synthetic operation. The properties of our macrocycles are discussed with respect to their potential to bind to biological targets that are not druggable by conventional, drug-like compounds. As an application of these artificial macrocycles we highlight potent p53-MDM2 antagonism.

Chelate Ring Size Effect as a Factor of Selective Fluorescent Recognition of Zn(2+) Ions by Pyrrolo[2,3-b]quinoxaline with a Substituted 2-Pyridyl Group Receptor.

Analysis of the spectral properties and structural differences of two turn-on ratiometric fluorescent receptors for Zn(2+) and Cd(2+) ions, derivatives of pyrrolo[2,3-b]quinoxaline (2), and earlier published 3 (Ostrowska et al. CrystEngComm 2015, 17, 498-502) was performed. Both ligands are E/Z push-pull olefins interconverting at room temperature, with barriers to rotation about enamine double bonds, from E to Z isomers of 19.3 ± 0.1 and 16.9 ± 0.3 kcal/mol and from Z to E of 16.9 ± 0.3 and 15.7 ± 0.2 kcal/mol, respectively. Diastereoisomers (E)-2 and (Z)-2 were isolated and characterized by X-ray structural analysis. The formation of complexes by (E/Z)-2 with acetates and acetylacetonates of Zn(2+) and Cd(2+) was monitored by UV-vis, fluorescence, and (1)H NMR titrations in acetonitrile, respectively. X-ray structural analysis for isolated [(E)-2]2Zn in relation to earlier published (E)-3-ZnOAc revealed the formation of a six-coordinated zinc ion with six- and four-membered bis-chelate rings by (E)-2. The chelate effect increases the ligand affinity for Zn(2+) (log β12 = 12.45) and causes the elongation of nitrogen-metal bonds. Extension of the coordination cavity size allows coordination of a cadmium ion. The introduction of a flexible ethylene linker between the fluorophore and ionophore pyridyl groups in 3 significantly affects the selectivity of zinc-ion recognition. The distorted tetrahedral geometry of (E)-3-ZnOAc with a four-coordinated zinc ion appears to be the most preferred because of the short donor-zinc distance with a 1:1 binding mode. The formation of the small coordination cavity size with six-membered bis-chelate rings provides an effective overlap of zinc and donor orbitals, precluding the coordination of a cadmium ion in the same manner as zinc.

Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of the MDM2-p53 interaction.

Using the computational pharmacophore-based ANCHOR.QUERY platform a new scaffold was discovered. Potent compounds evolved inhibiting the protein-protein interaction p53-MDM2. An extensive SAR study was performed based on our four-point pharmacophore model, yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using both fluorescence polarization (FP) assay and 2D-NMR-HSQC experiments.

Bioactive Macrocyclic Inhibitors of the PD-1/PD-L1 Immune Checkpoint

Blockade of the immunoinhibitory PD-1/PD-L1 pathway using monoclonal antibodies has shown impressive results with durable clinical antitumor responses. Anti-PD-1 and anti-PD-L1 antibodies have now been approved for the treatment of a number of tumor types, whereas the development of small molecules targeting immune checkpoints lags far behind. We characterized two classes of macrocyclic-peptide inhibitors directed at the PD-1/PD-L1 pathway. We show that these macrocyclic compounds act by directly binding to PD-L1 and that they are capable of antagonizing PD-L1 signaling and, similarly to antibodies, can restore the function of T-cells. We also provide the crystal structures of two of these small-molecule inhibitors bound to PD-L1. The structures provide a rationale for the checkpoint inhibition by these small molecules, and a description of their small molecule/PD-L1 interfaces provides a blueprint for the design of small-molecule inhibitors of the PD-1/PD-L1 pathway.

Scaffold hopping via ANCHOR.QUERY: β-lactams as potent p53-MDM2 antagonists

Using the pharmacophore-based virtual screening platform ANCHOR.QUERY, we morphed our recently described Ugi-4CR scaffold towards a β-lactam scaffold with potent p53-MDM2 antagonizing activities. 2D-HSQC and FP measurements confirm potent MDM2 binding. Molecular modeling studies are used to understand the observed SAR in the β-lactam series.

Conformations and Conformational Processes of Hexahydrobenzazocines by NMR and DFT Studies.

Conformational processes that occur in hexahydrobenzazocines have been studied with the (1)H and (13)C dynamic nuclear magnetic resonance (DNMR) spectroscopy. The coalescence effects are assigned to two different conformational processes: the ring-inversion of the ground-state conformations and the interconversion between two different conformers. The barriers for these processes are in the range of 42-52 and 42-43 kJ mol(-1), respectively. Molecular modeling on the density functional theory (DFT) level and the gauge invariant atomic orbitals (GIAO)-DFT calculations of isotropic shieldings and coupling constants for the set of low-energy conformations were compared with the experimental NMR data. The ground-state of all compounds in solution is the boat-chair (BC) conformation. The BC form adopts two different conformations because the nitrogen atom can be in the boat or chair parts of the BC structure. These two conformers are engaged in the interconversion process.

Artificial Macrocycles as Potent p53–MDM2 Inhibitors

Based on a combination of an Ugi four component reaction and a ring closing metathesis, a library of novel artificial macrocyclic inhibitors of the p53–MDM2 interaction was designed and synthesized. These macrocycles, alternatively to stapled peptides, target for the first time the large hydrophobic surface area formed by Tyr67, Gln72, His73, Val93, and Lys94 yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding affinity with MDM2 was evaluated using fluorescence polarization (FP) assay and 1H–15N two-dimensional HSQC nuclear magnetic resonance experiments.