Feasibility Studies for small molecule inhibitors targeting Neuropilin-1 in glioma


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Stella Tsirka and John Haley

Our overall goal is to conduct hit validation and hit-to-lead studies on our new small-molecule Neuropilin 1 (NRP1) antagonists as immune activators for cancer therapy. We have reported that genetic ablation of NRP1 in microglia and macrophages (MG/MP) improves outcomes in mouse models of primary glioma, i.e. decreases tumor volumes, vascularization, and the num- ber of immunosuppressive regulatory T cells (Tregs), and increases the number of proinflamma- tory MG/MPs and infiltration of cytotoxic CD8+ T cells. We have also reported that the prototype molecule EG00229 inhibits Nrp1 in vivo and reduces glioma growth when delivered by local in- fusion. Our endpoint in this project will be robust lead molecules suitable for final lead optimisa- tion and clinical candidate selection. We will also optimise our in vivo models of primary and metastatic brain tumors.

We started by identifying sub-mM peptidomimetic leads that did not have good oral bioavail- ability, and then followed this up by conducting a fragment screen and partly optimizing several hits with good potential for oral bioavailability. By incorporating these drug-like fragments into the design paradigm defined by EG00229, and a follow-up molecule EG01377, we will have generated candidate orally-deliverable, brain-penetrant inhibitors. For our team here at SBU, we now need to experimentally determine their potency, oral absorption and CNS penetration. The following goals will be pursued in this proposal:

Aim 1. Development of Structure-Activity Relationships (SAR), cell toxicity evaluation and blockade of TGFβ signalling with prototype molecules.

Rationale: TGFβ signalling modulates the immune microenvironment at sites of tumor.

Approach: Through fragment screen data and systematic investigation of chemotypes designed to bind in the NRP1 binding pocket, we have identified CNS penetrant, likely orally bioavailable, tight-binding inhibitors. These hits will be validated through resynthesis and detailed analysis (1H, 13C NMR, LCMS). Cell toxicity will be assessed by screening against primary mouse mac- rophages and Tregs, as well as for peripheral blood Treg cells and/or monocytes. The hits will be incubated with T cells, Tregs and MG/MPs and the effects on TGFβ signalling assessed as an immune functional readout, using ELISA assays for TGFβ and immunoblots for the levels of phosphorylated SMAD2/3 (downstream effectors of NRP1/TGFβR activation). These experi- ments will help define and establish the relationships between ligand structure and NRP1 activi- ty in the context of cells of the innate and adoptive immune response.

Aim 2. Development of fragment screen hits with potential for oral absorption and im- mune activity

Rationale: Ensure that the small molecules have good potency and stability prior to in vivo ap- plication.

Approach: Hits may be chemically modified according to our knowledge base of NRP1 to in- clude additional binding groups and improve affinity. Dose-response data in Surface Plasmon Resonance (SPR) and competitive fluorescent polarization (FP) assays will be used to triage the compounds. The best modified hits will be screened in in vitro absorption, distribution, me- tabolism and excretion (ADME) assays; S9 fraction (mouse, human) and parallel artificial mem- brane permeability assay (PAMPA) will serve as a surrogate for membrane permeability. After determining toxicity in vivo, we will establish gliomas in animals, deliver the compounds locally into the tumor or systemically by gavage, and compare them to the prototype EG00229 by measuring tumor volumes, infiltration of T lymphocytes, abundance of Tregs, and activation states of MG/MP.

Taken together, we aim to identify authentic orally-absorbed and CNS-penetrant small molecule inhibitors of NRP1 with nanomolar potency that can be used as immunomodulators of the tumor microenvironment.


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