Potency-Enhanced Peptidomimetic VHL Ligands with Improved Oral Bioavailability.

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity. Moreover, incorporating conformationally constrained alterations on the methylthiazole benzylamine moiety led to the development of highly potent VHL ligands with picomolar binding affinity and significantly improved oral bioavailability. We anticipate that our optimized VHL ligand, GNE7599, will serve as a valuable tool compound for investigating the VHL pathway and advancing the field of targeted protein degradation.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

Structures of the ApoL1 and ApoL2 N-terminal domains reveal a non-classical four-helix bundle motif.

Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.

Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Ribosomal Synthesis of Macrocyclic Peptides with Linear γ4- and ß-Hydroxy-γ4-amino Acids.

Herein, we report the ribosomal elongation of linear γ4- and ß-hydroxy-γ4-amino acids (statines) to expand the nonproteinogenic amino acid repertoire of natural product-like combinatorial peptide libraries. First, we demonstrated the successful ribosomal incorporation of four linear γ4-amino acids (γ4Gly, (S)-γ4Ala, (S)-γ4Nva, and (R)-γ4Leu) into a 10-mer macrocyclic peptide scaffold. Given the promising effects reported for statines on the cell permeability of macrocyclic peptides, we also designed and tested the ribosomal incorporation of six statines derived from Ala and d-val. Four Ala-derived statines were successfully incorporated into peptides, and γ4SAla3R-OH (GP2) showed a similar efficiency of incorporation to that of (S)-ß2hAla and l-Ala. These new building blocks might confer the important pharmacological properties of protease resistance and membrane permeability to macrocyclic peptides and expand the diversity of future combinatorial peptide libraries that can be translated by the ribosome.

Ribosomal Synthesis of Macrocyclic Peptides with ß2- and ß2,3-Homo-Amino Acids for the Development of Natural Product-Like Combinatorial Libraries.

The development of large, natural-product-like, combinatorial macrocyclic peptide libraries is essential in the quest to develop therapeutics for "undruggable" cellular targets. Herein we report the ribosomal synthesis of macrocyclic peptides containing one or more ß2-homo-amino acids (ß2haa) to enable their incorporation into mRNA display-based selection libraries. We confirmed the compatibility of 14 ß2-homo-amino acids, (S)- and (R)-stereochemistry, for single incorporation into a macrocyclic peptide with low to high translation efficiency. Interestingly, N-methylation of the backbone amide of ß2haa prevented the incorporation of this amino acid subclass by the ribosome. Additionally, we designed and incorporated several α,ß-disubstituted ß2,3-homo-amino acids (ß2,3haa) with different R-groups on the α- and ß-carbons of the same amino acid. Incorporation of these ß2,3haa enables increased diversity in a single position of a macrocyclic peptide without significantly increasing the overall molecular weight, which is an important consideration for passive cell permeability. We also successfully incorporated multiple (S)-ß2hAla into a single macrocycle with other non-proteinogenic amino acids, confirming that this class of ß-amino acid is suitable for development of large scale macrocyclic peptide libraries.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Heterobifunctional Molecules Induce Dephosphorylation of Kinases-A Proof of Concept Study.

Heterobifunctional molecules have proven powerful tools to induce ligase-dependent ubiquitination of target proteins. We describe here a chemical strategy for controlling a different post-translational modification (PTM): phosphorylation. Heterobifunctional molecules were designed to promote the proximity of a protein phosphatase (PP1) to protein targets. The synthesized molecules induced the PP1-dependent dephosphorylation of AKT and EGFR. To our knowledge, this work represents the first examples of small molecules recruiting non-native partners to induce removal of a PTM.

Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation.

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family.

Cyclodextrin Reduces Intravenous Toxicity of a Model Compound.

Solubilization of new chemical entities for toxicity assessment must use excipients that do not negatively impact drug pharmacokinetics and toxicology. In this study, we investigated the tolerability of a model freebase compound, GDC-0152, solubilized by pH adjustment with succinic acid and complexation with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) to enable intravenous use. Solubility, critical micelle concentration, and association constant with HP-ß-CD were determined. Blood compatibility and potential for hemolysis were assessed in vitro. Local tolerability was assessed after intravenous and subcutaneous injections in rats. A pharmacokinetic study was conducted in rats after intravenous bolus administration. GDC-0152 exhibited pH-dependent solubility that was influenced by self-association. The presence of succinic acid increased solubility in a concentration-dependent manner. HP-ß-CD alone also increased solubility, but the extent of solubility enhancement was significantly lower than succinic acid alone. Inclusion of HP-ß-CD in the solution of GDC-0152 improved blood compatibility, reduced hemolytic potential by ∼20-fold in vitro, and increased the maximum tolerated dose to 80 mg/kg.

Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9.

The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity-a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.

Publisher Correction: A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

The version of this article originally published contained older versions of the Life Sciences Reporting Summary and the Supplementary Text and Figures. The error has been corrected in the HTML and PDF versions of the article.

A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

Found in Translation: How Preclinical Research Is Guiding the Clinical Development of the BCL2-Selective Inhibitor Venetoclax.

Since the discovery of apoptosis as a form of programmed cell death, targeting the apoptosis pathway to induce cancer cell death has been a high-priority goal for cancer therapy. After decades of effort, drug-discovery scientists have succeeded in generating small-molecule inhibitors of antiapoptotic BCL2 family proteins. Innovative medicinal chemistry and structure-based drug design, coupled with a strong fundamental understanding of BCL2 biology, were essential to the development of BH3 mimetics such as the BCL2-selective inhibitor venetoclax. We review a number of preclinical studies that have deepened our understanding of BCL2 biology and facilitated the clinical development of venetoclax.Significance: Basic research into the pathways governing programmed cell death have paved the way for the discovery of apoptosis-inducing agents such as venetoclax, a BCL2-selective inhibitor that was recently approved by the FDA and the European Medicines Agency. Preclinical studies aimed at identifying BCL2-dependent tumor types have translated well into the clinic thus far and will likely continue to inform the clinical development of venetoclax and other BCL2 family inhibitors. Cancer Discov; 7(12); 1376-93. ©2017 AACR.

BCL2 mutations do not confer adverse prognosis in follicular lymphoma patients treated with rituximab.

BCL2 mutations have been suggested to confer an adverse prognosis to follicular lymphoma (FL) patients, but their prognostic value has not been assessed in patients treated with a rituximab-containing regimen. Here we evaluated the prognostic value of BCL2 mutations in a large prospective cohort of 252 patients with FL treated with immunochemotherapy in the PRIMA randomized trial. Using a DNA-targeted sequencing approach, we detected amino acid altering mutations in 135 patients (54%) and showed that these mutations were probably mediated by the over-activation of AICDA (activation-induced cytidine deaminase) in the context of the t(14;18) translocation. The BCL2 variants identified in PRIMA patients affected the BH1, BH2, and BH3 functional motifs at a lower frequency than the N-terminus and flexible loop domain, with mostly conservative aminoacid changes. With a median follow-up of 6.7 years, we did not observe any impact of BCL2 mutations either on overall survival or progression-free survival.

From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors.

Members of the B cell lymphoma 2 (BCL-2) gene family have a central role in regulating programmed cell death by controlling pro-apoptotic and anti-apoptotic intracellular signals. In cancer, apoptosis evasion through dysregulation of specific BCL-2 family genes is a recurring event; accordingly, selective inhibition of specific anti-apoptotic BCL-2 family proteins represents an exciting therapeutic opportunity. A combination of nuclear magnetic resonance (NMR)-based screening and structure-based drug design has yielded the first bona fide BCL-2 homology 3 (BH3) mimetics, including the BCL-2 and BCL-XL dual antagonist navitoclax, which is the first BCL-2 family inhibitor to show efficacy in patients with cancer. Clinical experience with navitoclax prompted the generation of the highly selective BCL-2 inhibitor venetoclax, which is now approved in the United States for the treatment of patients with chronic lymphocytic leukaemia with 17p deletion who have received at least one prior therapy. Recent advances have also been made in the development of potent and selective inhibitors of BCL-XL and myeloid cell leukaemia 1 (MCL1), which are additional BCL-2 family members with established anti-apoptotic roles in cancer. Here we review the latest progress in direct and selective targeting of BCL-2 family proteins for cancer therapy.

A Phase I Dose-Escalation Study Evaluating the Safety Tolerability and Pharmacokinetics of CUDC-427, a Potent, Oral, Monovalent IAP Antagonist, in Patients with Refractory Solid Tumors.

PURPOSE: To determine the dose-limiting toxicities (DLT), adverse events (AE), pharmacokinetics, and preliminary evidence of antitumor activity of CUDC-427 (formerly GDC-0917), a selective antagonist of inhibitor of apoptosis (IAP) proteins. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were treated with escalating doses of CUDC-427 orally on a daily 14-day on/7-day off schedule in 21-day cycles using a modified continuous reassessment method design. Blood samples were assayed to determine the pharmacokinetic properties, pharmacodynamic alterations of cellular IAP levels in peripheral blood mononuclear cells (PBMC), and monocyte chemoattractant protein-1 (MCP-1) levels. RESULTS: Forty-two patients received 119 cycles of CUDC-427. Overall, the most common treatment-related toxicities were fatigue, nausea, vomiting, and rash. One DLT (grade 3 fatigue) occurred in a patient at 450 mg dose level during cycle 1, and 5 patients experienced AEs related to CUDC-427 that led to discontinuation and included grade 3 pruritus, and fatigue, and grade 2 drug hypersensitivity, pneumonitis, rash, and QT prolongation. The maximum planned dose of 600 mg orally daily for 2 weeks was reached, which allometrically scaled to exceed the IC90 in preclinical xenograft studies. Significant decreases in cIAP-1 levels in PBMCs were observed in all patients 6 hours after initial dosing. Responses included durable complete responses in one patient with ovarian cancer and one patient with MALT lymphoma. CONCLUSIONS: CUDC-427 can be administered safely at doses up to 600 mg daily for 14 days every 3 weeks. The absence of severe toxicities, inhibition of cIAP-1 in PBMC, and antitumor activity warrant further studies. Clin Cancer Res; 22(18); 4567-73. ©2016 AACR.

Expression Profile of BCL-2, BCL-XL, and MCL-1 Predicts Pharmacological Response to the BCL-2 Selective Antagonist Venetoclax in Multiple Myeloma Models.

BCL-2 family proteins dictate survival of human multiple myeloma cells, making them attractive drug targets. Indeed, multiple myeloma cells are sensitive to antagonists that selectively target prosurvival proteins such as BCL-2/BCL-XL (ABT-737 and ABT-263/navitoclax) or BCL-2 only (ABT-199/GDC-0199/venetoclax). Resistance to these three drugs is mediated by expression of MCL-1. However, given the selectivity profile of venetoclax it is unclear whether coexpression of BCL-XL also affects antitumor responses to venetoclax in multiple myeloma. In multiple myeloma cell lines (n = 21), BCL-2 is expressed but sensitivity to venetoclax correlated with high BCL-2 and low BCL-XL or MCL-1 expression. Multiple myeloma cells that coexpress BCL-2 and BCL-XL were resistant to venetoclax but sensitive to a BCL-XL-selective inhibitor (A-1155463). Multiple myeloma xenograft models that coexpressed BCL-XL or MCL-1 with BCL-2 were also resistant to venetoclax. Resistance to venetoclax was mitigated by cotreatment with bortezomib in xenografts that coexpressed BCL-2 and MCL-1 due to upregulation of NOXA, a proapoptotic factor that neutralizes MCL-1. In contrast, xenografts that expressed BCL-XL, MCL-1, and BCL-2 were more sensitive to the combination of bortezomib with a BCL-XL selective inhibitor (A-1331852) but not with venetoclax cotreatment when compared with monotherapies. IHC of multiple myeloma patient bone marrow biopsies and aspirates (n = 95) revealed high levels of BCL-2 and BCL-XL in 62% and 43% of evaluable samples, respectively, while 34% were characterized as BCL-2(High)/BCL-XL (Low) In addition to MCL-1, our data suggest that BCL-XL may also be a potential resistance factor to venetoclax monotherapy and in combination with bortezomib. Mol Cancer Ther; 15(5); 1132-44. ©2016 AACR.