Brent Douty

Discovery of potent competitive inhibitors of indoleamine 2,3-dioxygenase with in vivo pharmacodynamic activity and efficacy in a mouse melanoma model.

A hydroxyamidine chemotype has been discovered as a key pharmacophore in novel inhibitors of indoleamine 2,3-dioxygenase (IDO). Optimization led to the identification of 5l, which is a potent (HeLa IC(50) = 19 nM) competitive inhibitor of IDO. Testing of 5l in mice demonstrated pharmacodynamic inhibition of IDO, as measured by decreased kynurenine levels (>50%) in plasma and dose dependent efficacy in mice bearing GM-CSF-secreting B16 melanoma tumors.

Structural Insights into the Design of Nonpeptidic Isothiazolidinone-containing Inhibitors of Protein-tyrosine Phosphatase 1B

Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp48, and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp48 and the backbone NH of Arg47 via a water molecule. Although both substituents retain the favorable hydrogen bonding network of the peptide scaffold, their aryl rings interact weakly with the protein. The aryl ring of benzimidazole is partially solvent exposed and only participates in van der Waals interactions with Phe182 of the flap. The aryl ring of aryl sulfonamide adopts an unexpected conformation and only participates in intramolecular π-stacking interactions with the benzimidazole ring. These results explain the flat SAR for substitutions on both rings and the reason why unsubstituted moieties were selected as candidates. Finally, substituents ortho to the IZD heterocycle on the aryl ring of the IZD-phenyl moiety bind in a small narrow site adjacent to the primary phosphate binding pocket. The crystal structure of an o-chloro derivative reveals that chlorine interacts extensively with residues in the small site. The structural insights that have led to the discovery of potent benzimidazole aryl sulfonamide o-substituted derivatives are discussed in detail.

Structural Basis for Inhibition of Protein-Tyrosine Phosphatase 1B by Isothiazolidinone Heterocyclic Phosphonate Mimetics.

Crystal structures of protein-tyrosine phosphatase 1B in complex with compounds bearing a novel isothiazolidinone (IZD) heterocyclic phosphonate mimetic reveal that the heterocycle is highly complementary to the catalytic pocket of the protein. The heterocycle participates in an extensive network of hydrogen bonds with the backbone of the phosphate-binding loop, Phe182 of the flap, and the side chain of Arg221. When substituted with a phenol, the small inhibitor induces the closed conformation of the protein and displaces all waters in the catalytic pocket. Saturated IZD-containing peptides are more potent inhibitors than unsaturated analogs because the IZD heterocycle and phenyl ring directly attached to it bind in a nearly orthogonal orientation with respect to each other, a conformation that is close to the energy minimum of the saturated IZD-phenyl moiety. These results explain why the heterocycle is a potent phosphonate mimetic and an ideal starting point for designing small nonpeptidic inhibitors.

INCB24360 (Epacadostat), a Highly Potent and Selective Indoleamine-2,3-dioxygenase 1 (IDO1) Inhibitor for Immuno-oncology

A data-centric medicinal chemistry approach led to the invention of a potent and selective IDO1 inhibitor 4f, INCB24360 (epacadostat). The molecular structure of INCB24360 contains several previously unknown or underutilized functional groups in drug substances, including a hydroxyamidine, furazan, bromide, and sulfamide. These moieties taken together in a single structure afford a compound that falls outside of “drug-like” space. Nevertheless, the in vitro ADME data is consistent with the good cell permeability and oral bioavailability observed in all species (rat, dog, monkey) tested. The extensive intramolecular hydrogen bonding observed in the small molecule crystal structure of 4f is believed to significantly contribute to the observed permeability and PK. Epacadostat in combination with anti-PD1 mAb pembrolizumab is currently being studied in a phase 3 clinical trial in patients with unresectable or metastatic melanoma.

Abstract C103: The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models

Understanding the in vivo responses to immunoregulatory agents provides a basis for building more efficacious combination regimens. Pharmacologic inhibition of the oncogenic PI3Kδ pathway has been shown to be active in patients with hematopoietic malignancies. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, through the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses, suggesting that it may have additional utility in this patient population. We identified a similar immunomodulatory role for the PI3Kδ-selective inhibitor INCB050465 in a preclinical model of pancreatic cancer, where an increase in the number of CD8+ T cells, a decrease in the number of suppressor cells and efficacy were seen. Therefore we explored the potential of INCB050465 in additional preclinical solid tumor models, alone and in combination with other immunotherapeutic agents. INCB050465 inhibited tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition was not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate immune-mediated mechanisms, tumors were analyzed for modulation of gene expression and immune phenotype after mice received short-term treatment. INCB050465 was shown to significantly downregulate the T cell gene signature in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. As seen previously, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T-cell-inflamed model by reducing both Treg and M2 macrophages, which promotes re-activation of both CD4+ and CD8+ T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell response over time. In summary, pharmacological inhibition of PI3Kδ can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and complex cellular responses may provide rationale to identify individuals who may benefit from PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Liang-Chuan Wang, Michael Hansbury, Yue Zhang, Gengjie Yang, Timothy Burn, Paul Waeltz, Mark Rupar, Eddy Yue, Brent Douty, Thomas Maduskuie, Nikoo Falahatpisheh, Yun-long Li, Andrew Combs, Gregory Hollis, Reid Huber, Peggy Scherle. The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C103.

Pharmacological inactivation of PI3Kδ in the tumor microenvironment enhances efficacy of other immunotherapeutic agents

Pharmacological inhibition of the oncogenic PI3Kδ pathway has been shown to be efficacious in patients with hematopoietic malignancies. However, its therapeutic application in patients with solid tumors has not yet been tested. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, resulting from the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses. Therefore we explored the immunotherapeutic potential of our PI3Kδ-selective compound, INCB050465, in multiple preclinical tumor models. We demonstrate that INCB050465 can block tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition is not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate the immune-mediated mechanisms, tumors exposed to vehicle or INCB050465 were harvested and analyzed for modulation of gene expression and immune phenotype. INCB050465 was shown to significantly downregulate T cell gene signatures in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. In contrast, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. We next examined INCB050465 in combination with other immune modulators. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T cell-inflamed model by reducing both Treg and M2 macrophages, which then allowed the re-activation of both CD4 and CD8 T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell responses over time. In summary, inactivation of PI3Kδ with a pharmacological inhibitor can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and associated cellular responses may provide a rationale to identify individuals who may benefit most from PI3Kδ inhibitor-based immunotherapy combinations in the clinic.