Synthesis and Biochemical Evaluation of Monocarboxylic GRB2 SH2 Domain Inhibitors.

This protocol discloses the synthesis of monocarboxylic inhibitors with a macrocyclic peptide scaffold to bind with the GRB2 SH2 domain and disrupt the protein-protein interactions (PPIs) between GRB2 and phosphotyrosine-containing proteins.

Clinical Application of Individualized 3D-Printed Chest Wall Conformal Device in IMRT for Post-Mastectomy Breast Cancer.

Background Breast cancer is the most common malignant tumour in women. Radical mastectomy with postoperative radiotherapy is now the standard treatment for locally advanced breast cancer. Intensity-modulated radiotherapy (IMRT) has now been developed, which employs linear accelerators to deliver precise radiation to a tumour while minimizing the dose to surrounding normal tissue. It significantly improves the efficacy of breast cancer treatment. However, there are still some flaws that must be addressed. Objective To assess the clinical application of the three-dimensional (3D)-printed chest wall conformal device for breast cancer patients who need to be treated by chest wall intensity modulated radiotherapy (IMRT) after radical mastectomy. Methods The 24 patients were divided into three groups. During a computed tomography (CT) scan, patients in the study group were fixed by a 3D-printed chest wall conformal device, nothing in control group A, and a traditional 1-cm thick silica gel compensatory pad on the chest wall in control group B. The parameters of mean Dmax, Dmean, D2%, D50%, D98%, the conformity index (CI), and the homogeneity index (HI) of the planning target volume (PTV) are compared. Results The study group had the best dose uniformity (HI = 0.092) and the highest conformation (CI = 0.97), the worst in control group A (HI = 0.304, CI = 0.84). The mean Dmax, Dmean, and D2% of the study group were lower than control groups A and B (p<0.05). The mean D50% was higher than control group B (p<0.05), while the mean D98% was higher than control groups A and B (p<0.05). The mean Dmax, Dmean, D2%, and HI of control group A were higher than control group B (p<0.05), whereas the mean D98% and CI were lower than control group B (p<0.05). Conclusion By improving the efficacy of postoperative radiotherapy for breast cancer, using 3D-printed chest wall conformal devices may greatly improve the accuracy of repeating position fixation, increase the dose on the skin surface of the chest wall, optimise the dose distribution of the target area, and thus further reduce tumour recurrence and prolong patients' survival.

Common Structural Features of Hydrophobic α-Helical Hot Spots: Insights for the Design of Novel α-Helix Mimetics.

The binding conformations of α-helical hydrophobic hot spots are convergent into two spatial areas in protein-protein complex structures. The physical basis for convergence was disclosed, which allows the development of pharmacophore models for i/i + 4/i + 7 or i/i + 3/i + 4 α-helical hot spots. Further investigation revealed that this convergence of binding conformations is common among all hydrophobic hot spots regardless of their α-helical positions. This observation led to a streamlined generation of pharmacophore models for hydrophobic hot spots at any positions along the α-helix. These successfully evaluated pharmacophore models may be useful for designing novel α-helical hot spot mimetics.

Fragment hopping protocol for the design of small-molecule protein-protein interaction inhibitors.

Fragment-based ligand discovery (FBLD) is one of the most successful approaches to designing small-molecule protein-protein interaction (PPI) inhibitors. The incorporation of computational tools to FBLD allows the exploration of chemical space in a time- and cost-efficient manner. Herein, a computational protocol for the development of small-molecule PPI inhibitors using fragment hopping, a fragment-based de novo design approach, is described and a case study is presented to illustrate the efficiency of this protocol. Fragment hopping facilitates the design of PPI inhibitors from scratch solely based on key binding features in the PPI complex structure. This approach is an open system that enables the inclusion of different state-of-the-art programs and softwares to improve its performances.

New ZW4864 Derivatives as Small-Molecule Inhibitors for the ß-Catenin/BCL9 Protein-Protein Interaction.

A series of 1-(3-(2-amino-2-oxoethoxy)phenyl)piperidine-3-carboxamide derivatives was reported as new small-molecule ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) inhibitors. Compounds 17-21 were discovered to inhibit the ß-catenin/BCL9 PPI with K i = 0.85-2.7 µM. The effects of 21 on the ß-catenin/BCL9 PPI in cellular context were demonstrated by ß-catenin/BCL9 pull-down inhibition and dose-dependent suppression of Wnt/ß-catenin signal transactivation. Notably, compound 21 is more potent than ZW4864, a previously reported analogue, in modulating transcription and expression of ß-catenin target genes and suppressing survival of ß-catenin-dependent cancer cells. The cellular on-target efficacy of 21 was demonstrated by ß-catenin rescue experiments. Compound 21 represents a promising starting point for further optimization of ß-catenin/BCL9 PPI inhibitors.

Characterization of Hydrophilic α-Helical Hot Spots on the Protein-Protein Interaction Interfaces for the Design of α-Helix Mimetics.

The cooperativity index, Kc, was developed to examine the binding synergy between hot spots of the ligand-protein. For the first time, the convergence of the side-chain spatial arrangements of hydrophilic α-helical hot spots Thr, Tyr, Asp, Asn, Ser, Cys, and His in protein-protein interaction (PPI) complex structures was disclosed and quantified by developing novel clustering models. In-depth analyses revealed the driving force for the protein-protein binding conformation convergence of hydrophilic α-helical hot spots. This observation allows deriving pharmacophore models to design new mimetics for hydrophilic α-helical hot spots. A computational protocol was developed to search amino acid analogues and small-molecule mimetics for each hydrophilic α-helical hot spot. As a pilot study, diverse building blocks of commercially available nonstandard L-type α-amino acids and the phenyl ring-containing small-molecule fragments were obtained, which serve as a fragment collection to mimic hydrophilic α-helical hot spots for the improvement of binding affinity, selectivity, physicochemical properties, and synthesis accessibility of α-helix mimetics.

Synthesis and structural characterization of a monocarboxylic inhibitor for GRB2 SH2 domain.

A monocarboxylic inhibitor was designed and synthesized to disrupt the protein-protein interaction (PPI) between GRB2 and phosphotyrosine-containing proteins. Biochemical characterizations show compound 7 binds with the Src homology 2 (SH2) domain of GRB2 and is more potent than EGFR1068 phosphopeptide 14-mer. X-ray crystallographic studies demonstrate compound 7 occupies the GRB2 binding site for phosphotyrosine-containing sequences and reveal key structural features for GRB2-inhibitor binding. This compound with a -1 formal charge offers a new direction for structural optimization to generate cell-permeable inhibitors for this key protein target of the aberrant Ras-MAPK signaling cascade.

Discovery of an Orally Bioavailable Small-Molecule Inhibitor for the ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

Aberrant activation of Wnt/ß-catenin signaling is strongly associated with many diseases including cancer invasion and metastasis. Small-molecule targeting of the central signaling node of this pathway, ß-catenin, is a biologically rational approach to abolish hyperactivation of ß-catenin signaling but has been demonstrated to be a difficult task. Herein, we report a drug-like small molecule, ZW4864, that binds with ß-catenin and selectively disrupts the protein-protein interaction (PPI) between B-cell lymphoma 9 (BCL9) and ß-catenin while sparing the ß-catenin/E-cadherin PPI. ZW4864 dose-dependently suppresses ß-catenin signaling activation, downregulates oncogenic ß-catenin target genes, and abrogates invasiveness of ß-catenin-dependent cancer cells. More importantly, ZW4864 shows good pharmacokinetic properties and effectively suppresses ß-catenin target gene expression in the patient-derived xenograft mouse model. This study offers a selective chemical probe to explore ß-catenin-related biology and a drug-like small-molecule ß-catenin/BCL9 disruptor for future drug development.

Discovery of 1-Benzoyl 4-Phenoxypiperidines as Small-Molecule Inhibitors of the ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

Structure-based design and optimization were performed to develop small-molecule ß-catenin/B-cell lymphoma 9 (BCL9) inhibitors and improve their inhibitory activities. Compound ZL3138 with a novel 1-benzoyl 4-phenoxypiperidine scaffold was discovered to disrupt the ß-catenin/BCL9 protein-protein interaction (PPI) with a Ki of 0.96 µM in AlphaScreen competitive inhibition assays and displayed good selectivity for ß-catenin/BCL9 over ß-catenin/E-cadherin PPIs. The binding mode of new inhibitors was characterized by structure-activity relationship and site-directed mutagenesis studies. Protein pull-down assays indicate that this series of compounds directly binds with ß-catenin. Cellular target engagement and co-immunoprecipitation experiments demonstrate that ZL3138 binds with ß-catenin and disrupts the ß-catenin/BCL9 interaction without affecting the ß-catenin/E-cadherin interaction in living cells. Further cell-based studies show that ZL3138 selectively suppresses transactivation of Wnt/ß-catenin signaling, regulates transcription and expression of Wnt target genes, and inhibits the growth of Wnt/ß-catenin-dependent cancer cells.

Discovery of 2-(3-(3-Carbamoylpiperidin-1-yl)phenoxy)acetic Acid Derivatives as Novel Small-Molecule Inhibitors of the ß-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

The ß-catenin/B-cell lymphoma 9 (BCL9) protein-protein interaction (PPI) is a potential target for the suppression of hyperactive Wnt/ß-catenin signaling that is vigorously involved in cancer initiation and development. Herein, we describe the medicinal chemistry optimization of a screening hit to yield novel small-molecule inhibitors of the ß-catenin/BCL9 interaction. The best compound 30 can disrupt the ß-catenin/BCL9 interaction with a Ki of 3.6 µM in AlphaScreen competitive inhibition assays. Cell-based experiments revealed that 30 selectively disrupted the ß-catenin/BCL9 PPI, while leaving the ß-catenin/E-cadherin PPI unaffected, dose-dependently suppressed Wnt signaling transactivation, downregulated oncogenic Wnt target gene expression, and on-target selectively inhibited the growth of cancer cells harboring aberrant Wnt signaling. This compound with a new chemotype can serve as a lead compound for further optimization of inhibitors for ß-catenin/BCL9 PPI.

Preclinical evaluation of [225Ac]Ac-DOTA-TATE for treatment of lung neuroendocrine neoplasms.

PURPOSE: There is significant interest in the development of targeted alpha-particle therapies (TATs) for treatment of solid tumors. The metal chelator-peptide conjugate, DOTA-TATE, loaded with the ß-particle emitting radionuclide 177Lu ([177Lu]Lu-DOTA-TATE) is now standard care for neuroendocrine tumors that express the somatostatin receptor 2 (SSTR2) target. A recent clinical study demonstrated efficacy of the corresponding [225Ac]Ac-DOTA-TATE in patients that were refractory to [177Lu]Lu-DOTA-TATE. Herein, we report the radiosynthesis, toxicity, biodistribution (BD), radiation dosimetry (RD), and efficacy of [225Ac]Ac-DOTA-TATE in small animal models of lung neuroendocrine neoplasms (NENs). METHODS: [225Ac]Ac-DOTA-TATE was synthesized and characterized for radiochemical yield, purity and stability. Non-tumor-bearing BALB/c mice were tested for toxicity and BD. Efficacy was determined by single intravenous injection of [225Ac]Ac-DOTA-TATE into SCID mice-bearing human SSTR2 positive H727 and H69 lung NENs. RD was calculated using the BD data. RESULTS: [225Ac]Ac-DOTA-TATE was synthesized with 98% yield, 99.8% purity, and displayed 97% stability after 2 days incubation in human serum at 37 °C. All animals in the toxicity study appeared healthy 5 months post injection with no indications of toxicity, except that animals that received ≥111 kBq of [225Ac]Ac-DOTA-TATE had chronic progressive nephropathy. BD studies revealed that the primary route of elimination is by the renal route. RD calculations determined pharmacokinetics parameters and absorbed α-emission dosages from 225Ac and its daughters. For both tumor models, a significant tumor growth delay and time to experimental endpoint were observed following a single administration of [225Ac]Ac-DOTA-TATE relative to controls. CONCLUSIONS: These results suggest significant potential for the clinical translation of [225Ac]Ac-DOTA-TATE for lung NENs.

Direct targeting of ß-catenin in the Wnt signaling pathway: Current progress and perspectives.

Aberrant activation of the Wnt/ß-catenin signaling circuit is associated with cancer recurrence and relapse, cancer invasion and metastasis, and cancer immune evasion. Direct targeting of ß-catenin, the central hub in this signaling pathway, is a promising strategy to suppress the hyperactive ß-catenin signaling but has proven to be highly challenging. Substantial efforts have been made to discover compounds that bind with ß-catenin, block ß-catenin-mediated protein-protein interactions, and suppress ß-catenin signaling. Herein, we characterize potential small-molecule binding sites in ß-catenin, summarize bioactive small molecules that directly target ß-catenin, and review structure-based inhibitor optimization, structure-activity relationship, and biological activities of reported inhibitors. This knowledge will benefit future inhibitor development and ß-catenin-related drug discovery.

Inhibition of p53 DNA binding by a small molecule protects mice from radiation toxicity.

Transcription factors are attractive therapeutic targets that are considered non-druggable because they do not have binding sites for small drug-like ligands. We established a cell-free high-throughput screening assay to search for small molecule inhibitors of DNA binding by transcription factors. A screen was performed using p53 as a target, resulting in the identification of NSC194598 that inhibits p53 sequence-specific DNA binding in vitro (IC50 = 180 nM) and in vivo. NSC194598 selectively inhibited DNA binding by p53 and homologs p63/p73, but did not affect E2F1, TCF1, and c-Myc. Treatment of cells with NSC194598 alone paradoxically led to p53 accumulation and modest increase of transcriptional output owing to disruption of the MDM2-negative feedback loop. When p53 was stabilized and activated by irradiation or chemotherapy drug treatment, NSC194598 inhibited p53 DNA binding and induction of target genes. A single dose of NSC194598 increased the survival of mice after irradiation. The results suggest DNA binding by p53 can be targeted using small molecules to reduce acute toxicity to normal tissues by radiation and chemotherapy.

Targeting the Side-Chain Convergence of Hydrophobic α-Helical Hot Spots To Design Small-Molecule Mimetics: Key Binding Features for i, i + 3, and i + 7.

The conformational convergence of hydrophobic α-helical hot spots was revealed by analyzing α-helix-mediated protein-protein interaction (PPI) complex structures. The pharmacophore models were derived for hydrophobic α-helical hot spots at positions i, i + 3, and i + 7. These provide the foundation for designing generalizable scaffolds that can directly mimic the binding mode of the side chains of α-helical hot spots, offering a new class of small-molecule α-helix mimetics. For the first time, the protocol was developed to identify the PPI targets that have similar binding pockets, allowing evaluation of inhibitor selectivities between α-helix-mediated PPIs. The mimicry efficiency of the previously designed scaffold 1 was disclosed. The close positioning of this small molecule to the additional α-helical hot spots suggests that the decoration of this series of generalizable scaffolds can conveniently reach the binding pockets of additional α-helical hot spots to produce potent small-molecule inhibitors for α-helix-mediated PPIs.

Inhibition of ß-catenin/B cell lymphoma 9 protein-protein interaction using α-helix-mimicking sulfono-γ-AApeptide inhibitors.

The rational design of α-helix-mimicking peptidomimetics provides a streamlined approach to discover potent inhibitors for protein-protein interactions (PPIs). However, designing cell-penetrating long peptidomimetic scaffolds equipped with various functional groups necessary for interacting with large protein-binding interfaces remains challenging. This is particularly true for targeting ß-catenin/BCL9 PPIs. Here we designed a series of unprecedented helical sulfono-γ-AApeptides that mimic the binding mode of the α-helical HD2 domain of B Cell Lymphoma 9 (BCL9). Our studies show that sulfono-γ-AApeptides can structurally and functionally mimic the α-helical domain of BCL9 and selectively disrupt ß-catenin/BCL9 PPIs with even higher potency. More intriguingly, these sulfono-γ-AApeptides can enter cancer cells, bind with ß-catenin and disrupt ß-catenin/BCL9 PPIs, and exhibit excellent cellular activity, which is much more potent than the BCL9 peptide. Furthermore, our enzymatic stability studies demonstrate the remarkable stability of the helical sulfono-γ-AApeptides, with no degradation in the presence of pronase for 24 h, augmenting their biological potential. This work represents not only an example of helical sulfono-γ-AApeptides that mimic α-helix and disrupt protein-protein interactions, but also an excellent example of potent, selective, and cell-permeable unnatural foldameric peptidomimetics that disrupt the ß-catenin/BCL9 PPI. The design of helical sulfono-γ-AApeptides may lead to a new strategy to modulate a myriad of protein-protein interactions.

Optimization of Peptidomimetics as Selective Inhibitors for the ß-Catenin/T-Cell Factor Protein-Protein Interaction.

The ß-catenin/T-cell factor (Tcf) protein-protein interaction (PPI) plays a critical role in the ß-catenin signaling pathway which is hyperactivated in many cancers and fibroses. Based on compound 1, which was designed to target the Tcf4 G13ANDE17 binding site of ß-catenin, extensive structure-activity relationship studies have been conducted. As a result, compounds 53 and 57 were found to disrupt the ß-catenin/Tcf PPI with the Ki values of 0.64 and 0.44 µM, respectively, and exhibit good selectivity for ß-catenin/Tcf over ß-catenin/E-cadherin and ß-catenin/adenomatous polyposis coli (APC) PPIs. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS) cell viability assays revealed that 56, the ethyl ester of 53, was more potent than 53 in inhibiting viability of most of the Wnt/ß-catenin hyperactive cancer cells. Further cell-based studies indicated that 56 disrupted the ß-catenin/Tcf PPI without affecting the ß-catenin/E-cadherin and ß-catenin/APC PPIs, suppressed transactivation of Wnt/ß-catenin signaling in dose-dependent manners, and inhibited migration and invasiveness of Wnt/ß-catenin-dependent cancer cells.

Ruthenium-Catalyzed C-H Allylation of Alkenes with Allyl Alcohols via C-H Bond Activation in Aqueous Solution.

A robust Ru(II)-catalyzed C-H allylation of electron-deficient alkenes with allyl alcohols in aqueous solution is reported. This method provides a straightforward and efficient access to the synthetically useful 1,4-diene skeletons. With the assistance of the N-methoxycarbamoyl directing group, this allylation reaction features a broad substrate scope with good functional group tolerance, excellent regio- and stereoselectivity, absence of metal oxidants, water-tolerant solvents, and mild reaction conditions. The mechanistic studies indicate that the process of the reversible C-H bond ruthenation is assisted by acetate, and the rate-determining step is unlikely to be the step of C-H bond cleavage.

Rhodium(iii)-catalyzed C-H allylation of indoles with allyl alcohols via ß-hydroxide elimination.

An efficient Rh(iii)-catalyzed dehydrative C-H allylation of indoles with allyl alcohols via ß-hydroxide elimination under oxidant-free conditions has been developed. This method features very mild reaction conditions, excellent regioselectivity and stereoselectivity, and compatibility with various functional groups. In addition, the directing group can be removed under mild reaction conditions, which further underscores the synthetic utility of this method.

AKR1B10 activates diacylglycerol (DAG) second messenger in breast cancer cells.

Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of action. Herein we report that AKR1B10 activates lipid second messengers to stimulate cell proliferation. Our data showed that ectopic expression of AKR1B10 in breast cancer cells MCF-7 promoted lipogenesis and enhanced levels of lipid second messengers, including phosphatidylinositol bisphosphate (PIP2), diacylglycerol (DAG), and inositol triphosphate (IP3). In contrast, silencing of AKR1B10 in breast cancer cells BT-20 and colon cancer cells HCT-8 led to decrease of these lipid messengers. Qualitative analyses by liquid chromatography-mass spectrum (LC-MS) revealed that AKR1B10 regulated the cellular levels of total DAG and majority of subspecies. This in turn modulated the phosphorylation of protein kinase C (PKC) isoforms PKCδ (Thr505), PKCµ (Ser744/748), and PKCα/ßII (Thr638/641) and activity of the PKC-mediated c-Raf/MEK/ERK signaling cascade. A pan inhibitor of PKC (Go6983) blocked ERK1/2 activation by AKR1B10. In these cells, phospho-p90RSK, phospho-MSK, and Cyclin D1 expression was increased by AKR1B10, and pharmacological inhibition of the ERK signaling cascade with MEK1/2 inhibitors U0126 and PD98059 eradicated induction of phospho-p90RSK, phospho-MSK, and Cyclin D1. In breast cancer cells, AKR1B10 promoted the clonogenic growth and proliferation of breast cancer cells in two-dimension (2D) and three-dimension (3D) cultures and tumor growth in immunodeficient female nude mice through activation of the PKC/ERK pathway. These data suggest that AKR1B10 stimulates breast cancer cell growth and proliferation through activation of DAG-mediated PKC/ERK signaling pathway.

Rhodium-Catalyzed [4 + 1] Cyclization via C-H Activation for the Synthesis of Divergent Heterocycles Bearing a Quaternary Carbon.

The development of an efficient approach to construct fused polycyclic systems bearing a quaternary carbon center represents a great challenge to synthetic chemistry. Herein, we report a Rh(III)-catalyzed [4 + 1] annulation of propargyl alcohols with various heterocyclic scaffolds under an air atmosphere. Diverse fused heterocycles containing a quaternary carbon center were obtained in moderate to good yields. Additionally, this method features a high atom-economy, metal oxidant free, simple operation, and compatibility with various functional groups.