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.
Blockade of the PD-1/PD-L1 immune checkpoint pathway with monoclonal antibodies has provided significant advances in cancer treatment. The antibody-based immunotherapies carry a number of disadvantages such as the high cost of the antibodies, their limited half-life, and immunogenicity. Development of small-molecule PD-1/PD-L1 inhibitors that could overcome these drawbacks is slow because of the incomplete structural information for this pathway. The first chemical PD-1/PD-L1 inhibitors have been recently disclosed by Bristol-Myers Squibb. Here we present NMR and X-ray characterization for the two classes of these inhibitors. The X-ray structures of the PD-L1/inhibitor complexes reveal one inhibitor molecule located at the center of the PD-L1 homodimer, filling a deep hydrophobic channel-like pocket between two PD-L1 molecules. Derivatives of (2-methyl-3-biphenylyl)methanol exhibit the structures capped on one side of the channel, whereas the compounds based on [3-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-methylphenyl]methanol induce an enlarged interaction interface that results in the open "face-back" tunnel through the PD-L1 dimer.
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.
Cancer cells can avoid and suppress immune responses through activation of inhibitory immune checkpoint proteins, such as PD-1, PD-L1, and CTLA-4. Blocking the activities of these proteins with monoclonal antibodies, and thus restoring T cell function, has delivered breakthrough therapies against cancer. In this review, we describe the latest work on structural characterization of the checkpoint proteins, their interactions with cognate ligands and with therapeutic antibodies. Structures of the extracellular portions of these proteins reveal that they all have a similar modular structure, composed of small domains similar in topology to the domains found in antibodies. Structural basis for blocking the PD-1/PD-L1 interaction by small molecules is illustrated with the compound BMS-202 that binds to and induces dimerization of PD-L1.
Mitochondria-originating reactive oxygen species (ROS) control T cell receptor (TCR)-induced gene expression. Here, we show that TCR-triggered activation of ADP-dependent glucokinase (ADPGK), an alternative, glycolytic enzyme typical for Archaea, mediates generation of the oxidative signal. We also show that ADPGK is localized in the endoplasmic reticulum and suggest that its active site protrudes toward the cytosol. The ADPGK-driven increase in glycolytic metabolism coincides with TCR-induced glucose uptake, downregulation of mitochondrial respiration, and deviation of glycolysis toward mitochondrial glycerol-3-phosphate dehydrogenase(GPD) shuttle; i.e., a metabolic shift to aerobic glycolysis similar to the Warburg effect. The activation of respiratory-chain-associated GPD2 results in hyperreduction of ubiquinone and ROS release from mitochondria. In parallel, mitochondrial bioenergetics and ultrastructure are altered. Downregulation of ADPGK or GPD2 abundance inhibits oxidative signal generation and induction of NF-κB-dependent gene expression, whereas overexpression of ADPGK potentiates them.
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.
Protein targeting by the signal recognition particle (SRP) is universally conserved and starts with the recognition of a signal sequence in the context of a translating ribosome. SRP54 and FtsY, two multidomain proteins with guanosine triphosphatase (GTPase) activity, are the central elements of the SRP system. They have to coordinate the presence of a signal sequence with the presence of a vacant translocation channel in the membrane. For coordination the two GTPases form a unique, nearly symmetric heterodimeric complex in which the activation of GTP hydrolysis plays a key role for membrane insertion of substrate proteins. Recent results are integrated in an updated perception of the order of events in SRP-mediated protein targeting.
SIMIBI-class (named after the signal recognition particle, MinD, BioD) nucleotide-binding proteins appeared early in evolution 1 and contain GTPases, as well as ATPases, involved in the correct localization of cellular constituents. The MinD ATPase, as the central part of the Min system, regulates the determination of the cell division site in all bacterial species 2 . SRP-GTPases form a subfamily of the SIMIBI class, with only three members: the signal sequence-binding protein Ffh (SRP54 in Eukarya and Archaea), the SRP receptor FtsY (SR in Eukarya) and FlhF, which is involved in flagella biosynthesis [3][4][5] . They share the conserved NG domain, which contains two major additions to the conserved fold of small G proteins. First, an --element (I-box) is inserted in the effector region; second, the N domain, comprising four -helices, is attached to the N terminus of the G domain. SRP (Ffh together with the SRP RNA) and FtsY constitute the universally conserved co-translational protein-targeting machinery 6,7 . When bound to GTP, Ffh and FtsY form, through interactions between their NG domains 8,9 , a heterodimeric complex that regulates the transfer of a ribosome-nascent chain complex to a vacant translocon in the membrane with a series of conformational rearrangements 10,11 . The two GTPases share a composite active site between their G domains in which GTP hydrolysis is reciprocally activated 12 . The SRP RNA [13][14][15] and membrane lipids 16,17 play fundamental roles in activating the Ffh-FtsY GTPases. The recent structure of the SRP-FtsY complex, together with biochemical implications, suggest that the distal end of the hairpin-like SRP RNA may be involved in this activation 18 . The third SRP-GTPase FlhF, together with the MinD-type protein YlxH (also known as FlhG, FleN, motR or MinD2), is essential for the placement and assembly of flagella 19 in many polar and peritrichous flagellated bacteria [20][21][22][23][24] . FlhF is required for the targeting of the first flagellar protein, FliF, to the cell pole 25 by a mechanism that is so far poorly understood. FlhF is associated with the membrane 25,26 and localizes at the cell pole 20 . The FlhF protein (Fig. 1a) contains an N-terminal B domain that seems to be involved in FliF targeting 25 ; it shares the NG domain fold with the other two members of the SRP-GTPase subfamily. FlhF forms a stable homodimer with GTP and a composite active site that is basically identical to the active site of the Ffh-FtsY heterodimer 5 . In both the homo-and heterodimer, the two nucleotides are bound in a head-to-tail manner, with the -phosphate of one nucleotide interacting with the 3 -OH of the ribose moiety of the other. However, for the homo-and heterodimers formed by the three SRP-GTPases, the molecular mechanism of activation is still unknown. We set out to understand the activation of SRPGTPases by studying FlhF. RESULTS The SRP-GTPase FlhF is activated by YlxHAs FlhF (Fig. 1) forms a stable homodimer, and reciprocal activation has not been observed 5 , we reasoned ...
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