Biochemical and structural basis for the pharmacological inhibition of nuclear hormone receptor PPARγ by inverse agonists.

Recent studies have reported that the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.

Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPα.

Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies. Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment crystallizable; FcγR: Fcγ receptor; Ig: immunoglobulin; IND: investigational new drug; MDM⊘: monocyte-derived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe combined immunodeficiency; SIRP: signal-regulatory protein.

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition.

Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.