Streamlined Target Deconvolution Approach Utilizing a Single Photoreactive Chloroalkane Capture Tag.

Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.

Cancer Stem Cell (CSC) Inhibitors in Oncology-A Promise for a Better Therapeutic Outcome: State of the Art and Future Perspectives.

Cancer stem cells (CSCs), a subpopulation of cancer cells endowed with self-renewal, tumorigenicity, pluripotency, chemoresistance, differentiation, invasive ability, and plasticity, reside in specialized tumor niches and are responsible for tumor maintenance, metastasis, therapy resistance, and tumor relapse. The new-age "hierarchical or CSC" model of tumor heterogeneity is based on the concept of eradicating CSCs to prevent tumor relapse and therapy resistance. Small-molecular entities and biologics acting on various stemness signaling pathways, surface markers, efflux transporters, or components of complex tumor microenvironment are under intense investigation as potential anti-CSC agents. In addition, smart nanotherapeutic tools have proved their utility in achieving CSC targeting. Several CSC inhibitors in clinical development have shown promise, either as mono- or combination therapy, in refractory and difficult-to-treat cancers. Clinical investigations with CSC marker follow-up as a measure of clinical efficacy are needed to turn the "hype" into the "hope" these new-age oncology therapeutics have to offer.

Global PROTAC Toolbox for Degrading BCR-ABL Overcomes Drug-Resistant Mutants and Adverse Effects.

The BCR-ABL fusion oncoprotein causes chronic myeloid leukemia or acute lymphoblastic leukemia in Ph+ patients because the ABL kinase is constitutively activated. However, current clinical treatment with ABL inhibitors is seriously limited by drug resistance and adverse effects. Although the emerging proteolysis-targeting chimeras (PROTACs) have been introduced to degrade BCR-ABL, most of them showed limited activity and could not overcome the common drug-resistant mutants, especially for T315I mutant. Herein, we systematically designed a set of unique PROTACs by globally targeting all the three binding sites of BCR-ABL, including dasatinib-, ponatinib-, and asciminib-based PROTACs. Our ponatinib-based PROTACs showed practical activity as dasatinib-based PROTACs, while no reported ponatinib-based PROTACs could degrade BCR-ABL before. As a proof of concept, some additional dasatinib-based PROTACs were then designed to degrade T315I mutant too. We provided a global PROTAC toolbox for degrading both wild-type and T315I-mutated BCR-ABL from each binding site. More importantly, these PROTACs showed better selectivity and less adverse effects than the inhibitors, indicating that PROTACs had great potential for overcoming clinical drug resistance and safety issues.

First-in-Humans Trial of Dasatinib-Derivative Tracer for Tumor Kinase-Targeted PET.

We developed a first-of-kind dasatinib-derivative imaging agent, 18F-SKI-249380 (18F-SKI), and validated its use for noninvasive in vivo tyrosine kinase-targeted tumor detection in preclinical models. In this study, we assessed the feasibility of using 18F-SKI for PET imaging in patients with malignancies. Methods: Five patients with a prior diagnosis of breast cancer, renal cell cancer, or leukemia underwent whole-body PET/CT imaging 90 min after injection of 18F-SKI (mean, 241.24 ± 116.36 MBq) as part of a prospective study. In addition, patients underwent either a 30-min dynamic scan of the upper abdomen including, at least partly, cardiac left ventricle, liver, spleen, and kidney (n = 2) or three 10-min whole-body PET/CT scans (n = 3) immediately after injection and blood-based radioactivity measurements to determine the time course of tracer distribution and facilitate radiation dose estimates. A subset of 3 patients had a delayed whole-body PET/CT scan at 180 min. Biodistribution, dosimetry, and tumor uptake were quantified. Absorbed doses were calculated using OLINDA/EXM 1.0. Results: No adverse events occurred after injection of 18F-SKI. In total, 27 tumor lesions were analyzed, with a median SUVpeak of 1.4 (range, 0.7-2.3) and tumor-to-blood ratios of 1.6 (range, 0.8-2.5) at 90 min after injection. The intratumoral drug concentrations calculated for 4 reference lesions ranged from 0.03 to 0.07 nM. In all reference lesions, constant tracer accumulation was observed between 30 and 90 min after injection. A blood radioassay indicated that radiotracer clearance from blood and plasma was initially rapid (blood half-time, 1.31 ± 0.81 min; plasma, 1.07 ± 0.66 min; n = 4), followed variably by either a prolonged terminal phase (blood half-time, 285 ± 148.49 min; plasma, 240 ± 84.85 min; n = 2) or a small rise to a plateau (n = 2). Like dasatinib, 18F-SKI underwent extensive metabolism after administration, as evidenced by metabolite analysis. Radioactivity was predominantly cleared via the hepatobiliary route. The highest absorbed dose estimates (mGy/MBq) in normal tissues were to the right colon (0.167 ± 0.04) and small intestine (0.153 ± 0.03). The effective dose was 0.0258 mSv/MBq (SD, 0.0034 mSv/MBq). Conclusion:18F-SKI demonstrated significant tumor uptake, distinct image contrast despite low injected doses, and rapid clearance from blood.

Azo-PROTAC: Novel Light-Controlled Small-Molecule Tool for Protein Knockdown.

Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable azobenzene-proteolysis targeting chimeras (Azo-PROTACs) by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photoswitchability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off properties.

Design and synthesis of novel dasatinib derivatives as inhibitors of leukemia stem cells.

We used the concept of bioisosteres to design and synthesize a novel series of dasatinib derivatives for the treatment of leukemia. Unfortunately, most of the dasatinib derivatives did not show appreciable inhibition against leukemia cell lines K562 and HL60. However, acrylamide compound 2c had comparable inhibitory activity with dasatinib against K562 cells (IC50 = 0.039 nM vs. 0.069 nM). And amide compound 2a and acrylamide compound 2c also had comparable inhibitory activity with dasatinib against the leukemia cell line HL60 (IC50 = 0.25 nM and 0.26 nM vs. 0.11 nM). Against the leukemia progenitor cell line KG1a, triazole compounds 15a and 15d-15f and oxadiazole compounds 24a-24d were more potent than dasatinib. In particular, the hydroxyl compounds 15a and 24a were about 64 and 180 fold more potent than dasatinib against KG1a cells (IC50 = 0.14 µM and 0.05 µM vs. 8.98 µM). Compounds 15a and 24a also inhibited colony formation in MCF-7 cells and inhibited cell migration in the cell wound scratch assay in B16BL6 cells. Moreover, hydroxyl compounds 15a and 24a had low toxicity in vivo.

Emergence of a Promising Lead Compound in the Treatment of Triple Negative Breast Cancer: An Insight into Conformational Features and Ligand Binding Landscape of c-Src Protein with UM-164.

UM-164, a potent Src/p38 inhibitor, is a promising lead compound for developing the first targeted therapeutic strategy against triple-negative breast cancer (TNBC). However, lack of understanding of conformational features of UM-164 in complex with Src serves a challenge in the rational design of novel Src dual inhibitors. Herein, we provide an in-depth insight into conformational features of Src-UM-164 using different computational approaches. This involved molecular dynamics (MD) simulation, principal component analysis (PCA), thermodynamics calculations, dynamic cross-correlation (DCCM) analysis, and hydrogen bond formation. Findings from this study revealed that (1) the binding of UM-164 to Src induces a more stable and compact conformation; (2) the binding of UM-164 results in increased correlation among the active site residue; (3) the presence of multiple phenyl rings and fluorinated phenyl group in UM-164 contributes to the steric effect; (4) a relatively high-binding free energy estimated for the Src-UM-164 system is affirmative of its experimental potency; (5) hydrophobic packing contributes significantly to the drug binding in Src-UM-164; and (6) observed increase in H-bond distance of interacting residue atoms and Dasatinib compared to UM-164. Findings from this study can serve as a baseline in the design of novel Src inhibitors with dual inhibitory properties.

Synthesis and biological evaluation of novel dasatinib analogues as potent DDR1 and DDR2 kinase inhibitors.

Novel dasatinib analogues as DDR1 and DDR2 inhibitors were designed and synthesized. The synthesized compounds were screened for DDR1 and DDR2 kinase inhibitory and cancer cell proliferation inhibitory activities. Some of the compounds showed the potent inhibitory activities against both DDR1 and DDR2, as well as anticancer activity in low nanomolar range against K562 cell line; especially, compound 3j demonstrated significantly better inhibitory potency than the parental dasatinib against both DDRs and also demonstrated the potent inhibitory activity against K562 cell lines (IC50 values of 2.26±0.46 nm for DDR1, 7.04±2.90 nm for DDR2, and 0.125±0.017 nm for K562 cell line).

2-Aminoxazole and 2-Aminothiazole Dasatinib Derivatives as Potent Inhibitors of Chronic Myeloid Leukemia K562 Cells.

Dasatinib is an important drug against chronic myeloid leukemia (CML). In this paper, we describe the preparation and anti-CML activity of 2-aminoxazole and 2-aminothiazole dasatinib derivatives. Biological activity was measured by the inhibition of proliferation of human CML K562 cells. The 2-aminoxazole derivatives had similar activities as the 2-aminothiazole derivatives. All newly synthesized compounds demonstrated more potent antiproliferative activity than imatinib. A few compounds (8b, 8c, 9b) showed nanomolar inhibitory activity, similar to that of dasatinib.

Conformation-Selective Analogues of Dasatinib Reveal Insight into Kinase Inhibitor Binding and Selectivity.

In the kinase field, there are many widely held tenets about conformation-selective inhibitors that have yet to be validated using controlled experiments. We have designed, synthesized, and characterized a series of kinase inhibitor analogues of dasatinib, an FDA-approved kinase inhibitor that binds the active conformation. This inhibitor series includes two Type II inhibitors that bind the DFG-out inactive conformation and two inhibitors that bind the αC-helix-out inactive conformation. Using this series of compounds, we analyze the impact that conformation-selective inhibitors have on target binding and kinome-wide selectivity.