Krzysztof M. Zak

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.

Mdm2 and MdmX inhibitors for the treatment of cancer: a patent review (2011 – present)

Introduction: One of the hallmarks of cancer cells is the inactivation of the p53 pathway either due to mutations in the p53 gene or over-expression of negative regulators, Mdm2 and/or MdmX. Pharmacological disruption of the Mdm2/X–p53 interaction to restore p53 activity is an attractive concept, aiming at a targeted and non-toxic cancer treatment. Areas covered: The introduction covers the biological role of p53 pathway and its regulation by Mdm2 and MdmX in normal and cancer cells and the current repertoire and development status of inhibitors of the Mdm2/X–p53 interaction for the treatment of cancer. The main part of the article covers patents and patent applications describing small molecule inhibitors of the Mdm2/X–p53 interaction published from 2011 until 2012. Expert opinion: The area of small molecule Mdm2/X–p53 interaction inhibitor development is progressing fast. Several Phase I clinical studies and preclinical programs are now in progress, however, the clinical proof concept has yet to be demonstrated. Multiple available compounds inhibit Mdm2–p53 interaction with nanomolar affinities, but MdmX is still missing such potent binders. Since research points to a complementary mode of Mdm2 and MdmX action, the future compound classes will possibly want to include dual actions versus Mdm2 and MdmX.

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.

Inhibitors of programmed cell death 1 (PD-1): a patent review (2010-2015).

The search for the magic bullet in cancer immunotherapies is ongoing since more than 100 years, but only recent clinical success of immune checkpoint directed antibodies significantly revived the field of immune-oncology [1]. A key protein target in this area is the protein–protein interaction (PPI) between PD-1 and its ligand PD-L1. Functionally, PD-1, also called programmed death-1 protein, comprises an immune checkpoint located on T-cells. The PD-1/PD-L1 axis is hijacked by viruses and tumor/cancer cells to suppress the immune surveillance. For example, PD-L1 is expressed on tumor cells and also on immune cells (e.g. myeloid tumor-infiltrating cells). Binding of PD-1 to PD-L1 determines a downregulation of T-cell effector functions in cancer patients, inhibiting the antitumor immune response and leading to T-cell exhaustion [2]. In viral diseases, a similar mechanism is used by viruses to undermine the effective immune recognitions [3]. Current medication directed toward the PD-1/PD-L1 axis includes monoclonal antibodies. These have shown impressive clinical results in the treatment of several types of tumors, including melanoma and lung cancer [4]. Currently, two humanized monoclonal antibodies targeting PD-1 are approved by the regulatory bodies, pembrolizumab and nivolumab [5]. Multiple additional clinical trials either as single agents or in combination with other agents are ongoing to extend their indication areas. Therapeutic antibodies however exhibit several disadvantages such as limited tissue and tumor penetration, very long half-life time, lacking oral bioavailability, immunogenicity, and difficult and expensive production. Moreover, current PD1/PD-L1 axis directed monoclonal antibodies lead to a tumor response only in a fraction of cases and tumor types. Therefore, a search for non-mAbs, including small molecules, peptides, cyclo-peptides, and macrocycles is ongoing and will be reviewed here.

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.

2,30-Bis(10H-indole) heterocycles: New p53/MDM2/MDMX antagonists.

The protein–protein interaction of p53 and MDM2/X is a promising non genotoxic anticancer target. A rapid and efficient methodology was developed to synthesize the 2,30-bis(10H-indole) heterocyclic scaffold 2 as ester, acid and amide derivatives. Their binding affinity with MDM2 was evaluated using both fluorescence polarization (FP) assay and HSQC experiments, indicating good inhibition and a perfect starting point for further optimizations.

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.

CCDC 1054956: Experimental Crystal Structure Determination

Related Article: Ewa Surmiak, Aleksandra Twarda-Clapa, Krzysztof M. Zak, Bogdan Musielak, Marcin D. Tomala, Katarzyna Kubica, Przemyslaw Grudnik, Mariusz Madej, Mateusz Jablonski, Jan Potempa, Justyna Kalinowska-Tluscik, Alexander Domling, Grzegorz Dubin, Tad A. Holak|2016|ACS Chem.Biol.|11|3310|doi:10.1021/acschembio.6b00596