Discovery and characterization of allosteric CD45 inhibitors
Bibliographic record
Abstract
CD45 phosphatase is expressed ubiquitously on cells of lymphoid origin, and has long been considered a drug target for immunosuppression. CD45 is a key component of the signaling pathway in immune cells, regulating activation and development of these cells. Drug intervention could suppress growth and provide a therapeutic avenue for treatment of transplant rejection, auto-immune diseases such as rheumatoid arthritis, and various cancers of the immune system. The intracellular, catalytic portion of CD45 is comprised of the D1 and D2 domains. The active site is located in the D1 domain, and the D2 domain is catalytically inactive. We decided to target the inter-domain region between D1 and D2 for the development of a small molecule allosteric inhibitor. The linker region between CD45 D1 and D2 was modeled in silico, and NCI database was screened for compounds that dock in this region. Hits were tested in vitro and we detected two CD45 inhibitors, one of which was demonstrated to be highly selective for CD45 in tyrosine phosphatase counter-assays. Analogs were generated which increased in vitro potency, and cell membrane permeability. The most active inhibitor, 211, was pursued further. The kinetics of CD45 inhibition by 211 indicated a non-competitive and irreversible mechanism of action, as well as some degree of substrate specificity. Site-directed mutations in CD45 enzyme at the predicted 211 binding sites revealed a loss of inhibition by 211 in the mutant CD45, consistent with the in silico prediction of allosteric inhibition. Circular dichroism studies indicated a conformational change in CD45 enzyme, in the presence of 211. We first validated that CD45 was the target of 211 in cells, by evidence of a change in the phosphorylation of its in vivo substrate Lck in Jurkat cells. As a control, the phosphorylation status of Lck was not affected in J45.01 (CD45-negative) cells with 211 treatment. 211 was shown to suppress the activation of T cell receptor signaling in primary T cells. In vivo, 211 inhibits the antigen-induced immune response in the mouse footpad.CD45 inhibitor 211 was assayed for its anti-cancer properties in the EL4 mouse thymoma cell line. 211 inflicted dose-dependent toxicity on these cells via cell cycle rest and apoptosis. EL4 cells injected into mice were delayed in developing a solid tumor when mice were treated with 211, and metastasis to the lymph nodes was reduced in these animals. Our work has shown the therapeutic potential of targeting CD45 by revealing a novel allosteric binding site for a CD45 inhibitor, and this approach may be useful for targeting other two-domain tyrosine phosphatases.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".