Fragment-Based Drug Discovery Targeting Inhibitor of Apoptosis Proteins: Discovery of a Non-Alanine Lead Series with Dual Activity Against cIAP1 and XIAP

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein–ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X‑Linked Inhibitor of Apoptosis Protein (XIAP)

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology

Fragment-Based Approach to the Development of an Orally Bioavailable Lactam Inhibitor of Lipoprotein-Associated Phospholipase A2 (Lp-PLA₂)

Lp-PLA₂ has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA₂. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC₅₀ > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA₂ inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile

A Fragment-Derived Clinical Candidate for Antagonism of X‑Linked and Cellular Inhibitor of Apoptosis Proteins: 1‑(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl‑1<i>H</i>,2<i>H</i>,3<i>H</i>‑pyrrolo[3,2‑<i>b</i>]pyridin-1-yl)-2-[(2<i>R</i>,5<i>R</i>)‑5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660)

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP–XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate <b>27</b> (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423)

A Fragment-Derived Clinical Candidate for Antagonism of X‑Linked and Cellular Inhibitor of Apoptosis Proteins: 1‑(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl‑1<i>H</i>,2<i>H</i>,3<i>H</i>‑pyrrolo[3,2‑<i>b</i>]pyridin-1-yl)-2-[(2<i>R</i>,5<i>R</i>)‑5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660)

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP–XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate <b>27</b> (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423)

Structure of the Epigenetic Oncogene MMSET and Inhibition by <i>N</i>‑Alkyl Sinefungin Derivatives

The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of <i>N</i>-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET

Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2‑Related Factor 2 (KEAP1:NRF2) Protein–Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch–NRF2 interaction. X-ray crystallographic screening identified three distinct “hot-spots” for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and <i>in vivo</i> models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1–NRF2 interaction

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors