Discovery of bivalent small molecule degraders of cyclin-dependent kinase 7 (CDK7).

Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.

A Coordinating Small Organic Molecule with Tunable Lower Critical Solution Temperature for Efficient Management of Solar Radiation.

Structurally well-defined small molecules with lower critical solution temperature (LCST) behavior offer enormous prospects for fine-tuning their phase transition properties to be "on-demand" applied in the specific scene but are still underexplored. Herein, a novel amphiphilic small LCST molecule is rationally designed and synthesized. The molecule, namely TG, features a conjugation of multiple short ethylene glycol (EG) chains with the functional coordinating terpyridine (Tpy) moiety. The molecule TG demonstrates excellent LCST behavior down to 0.05 × 10-3 m in a water solution. And a cloud point Tcp = 30.9 °C with a very short thermal hysteresis ΔT = 0.2 °C and good reversibility can be achieved when c = 0.1 × 10-3 m. The excellent LCST properties of TG have enabled its successful performance as the smart window for solar radiation management with the ∆Tlum, ∆TIR, and ∆Tsol being 83.6%, 49.1%, and 67.2%, respectively. Moreover, the presence of Tpy moiety in TG enables its coordination with Ru3+ and the resulting complex also exhibits modulated LCST behavior with different concentration-dependent Tcp. These studies would provide novel small-molecule-based scaffolds for constructing better solar radiation management systems as well as other thermal-responsive smart materials.

Discovery of a Potent Degrader for Fibroblast Growth Factor Receptor 1/2.

Aberrant activation of FGFR signaling occurs in many cancers, and ATP-competitive FGFR inhibitors have received regulatory approval. Despite demonstrating clinical efficacy, these inhibitors exhibit dose-limiting toxicity, potentially due to a lack of selectivity amongst the FGFR family and are poorly tolerated. Here, we report the discovery and characterization of DGY-09-192, a bivalent degrader that couples the pan-FGFR inhibitor BGJ398 to a CRL2VHL E3 ligase recruiting ligand, which preferentially induces FGFR1&2 degradation while largely sparing FGFR3&4. DGY-09-192 exhibited two-digit nanomolar DC50 s for both wildtype FGFR2 and several FGFR2-fusions, resulting in degradation-dependent antiproliferative activity in representative gastric cancer and cholangiocarcinoma cells. Importantly, DGY-09-192 induced degradation of a clinically relevant FGFR2 fusion protein in a xenograft model. Taken together, we demonstrate that DGY-09-192 has potential as a prototype FGFR degrader.

Selective Degradation of GSPT1 by Cereblon Modulators Identified via a Focused Combinatorial Library.

Cereblon (CRBN) is an E3 ligase adapter protein that can be reprogrammed by imide-class compounds such as thalidomide, lenalidomide, and pomalidomide to induce the degradation of neo-substrate proteins. In order to identify additional small molecule CRBN modulators, we implemented a focused combinatorial library approach where we fused an imide-based CRBN-binding pharmacophore to a heterocyclic scaffold, which could be further elaborated. We screened the library for CRBN-dependent antiproliferative activity in the multiple myeloma cell line MM1.S and identified five hit compounds. Quantitative chemical proteomics of hit compounds revealed that they induced selective degradation of GSPT1, a translation termination factor that is currently being explored as a therapeutic target for the treatment of acute myeloid leukemia. Molecular docking studies with CRBN and GSPT1 followed by analogue synthesis identified a possible hydrogen bond interaction with the central pyrimidine ring as a molecular determinant of hit compounds' selectivity. This study demonstrates that a focused combinatorial library design, phenotypic screening, and chemical proteomics can provide a suitable workflow to efficiently identify novel CRBN modulators.

Structure and Characterization of a Covalent Inhibitor of Src Kinase.

Unregulated Src activity promotes malignant processes in cancer, but no Src-directed targeted therapies are used clinically, possibly because early Src inhibitors produce off-target effects leading to toxicity. Improved selective Src inhibitors may enable Src-directed therapies. Previously, we reported an irreversible Src inhibitor, DGY-06-116, based on the hybridization of dasatinib and a promiscuous covalent kinase probe SM1-71. Here, we report biochemical and biophysical characterization of this compound. An x-ray co-crystal structure of DGY-06-116: Src shows a covalent interaction with the kinase p-loop and occupancy of the back hydrophobic kinase pocket, explaining its high potency, and selectivity. However, a reversible analog also shows similar potency. Kinetic analysis shows a slow inactivation rate compared to other clinically approved covalent kinase inhibitors, consistent with a need for p-loop movement prior to covalent bond formation. Overall, these results suggest that a strong reversible interaction is required to allow sufficient time for the covalent reaction to occur. Further optimization of the covalent linker may improve the kinetics of covalent bond formation.

A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging.

Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.

Structure-Based Design of a Potent and Selective Covalent Inhibitor for SRC Kinase That Targets a P-Loop Cysteine.

SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P-loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) SM1-71 as a starting point, we developed covalent inhibitor 15a, which discriminates SRC from other covalent targets of SM1-71 including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound 15a exhibited sustained inhibition of SRC signaling both in vitro and in vivo. Moreover, 15a exhibited potent antiproliferative effects in nonsmall cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.

Small molecule degraders of the hepatitis C virus protease reduce susceptibility to resistance mutations.

Targeted protein degradation is a promising drug development paradigm. Here we leverage this strategy to develop a new class of small molecule antivirals that induce proteasomal degradation of viral proteins. Telaprevir, a reversible-covalent inhibitor that binds to the hepatitis C virus (HCV) protease active site is conjugated to ligands that recruit the CRL4CRBN ligase complex, yielding compounds that can both inhibit and induce the degradation of the HCV NS3/4A protease. An optimized degrader, DGY-08-097, potently inhibits HCV in a cellular infection model, and we demonstrate that protein degradation contributes to its antiviral activity. Finally, we show that this new class of antiviral agents can overcome viral variants that confer resistance to traditional enzymatic inhibitors such as telaprevir. Overall, our work provides proof-of-concept that targeted protein degradation may provide a new paradigm for the development of antivirals with superior resistance profiles.

Quinoline and thiazolopyridine allosteric inhibitors of MALT1.

Quinolines and thiazolopyridines were developed as allosteric inhibitors of MALT1, with good cellular potency and exquisite selectivity. Mouse pharmacokinetic (PK) profiling showed these to have low in vivo clearance, and moderate oral exposure. The thiazolopyridines were less lipophilic than the quinolines, and one thiazolopyridine example was active in our hIL10 mouse pharmacodynamic (PD) model upon oral dosing.

Peptide-based covalent inhibitors of MALT1 paracaspase.

Potent and selective substrate-based covalent inhibitors of MALT1 protease were developed from the tetrapeptide tool compound Z-VRPR-fmk. To improve cell permeability, we replaced one arginine residue. We further optimized a series of tripeptides and identified compounds that were potent in both a GloSensor reporter assay measuring cellular MALT1 protease activity, and an OCI-Ly3 cell proliferation assay. Example compounds showed good overall selectivity towards cysteine proteases, and one compound was selected for further profiling in ABL-DLBCL cells and xenograft efficacy models.

Leveraging Compound Promiscuity to Identify Targetable Cysteines within the Kinome.

Covalent kinase inhibitors, which typically target cysteine residues, represent an important class of clinically relevant compounds. Approximately 215 kinases are known to have potentially targetable cysteines distributed across 18 spatially distinct locations proximal to the ATP-binding pocket. However, only 40 kinases have been covalently targeted, with certain cysteine sites being the primary focus. To address this disparity, we have developed a strategy that combines the use of a multi-targeted acrylamide-modified inhibitor, SM1-71, with a suite of complementary chemoproteomic and cellular approaches to identify additional targetable cysteines. Using this single multi-targeted compound, we successfully identified 23 kinases that are amenable to covalent inhibition including MKNK2, MAP2K1/2/3/4/6/7, GAK, AAK1, BMP2K, MAP3K7, MAPKAPK5, GSK3A/B, MAPK1/3, SRC, YES1, FGFR1, ZAK (MLTK), MAP3K1, LIMK1, and RSK2. The identification of nine of these kinases previously not targeted by a covalent inhibitor increases the number of targetable kinases and highlights opportunities for covalent kinase inhibitor development.

A multitargeted probe-based strategy to identify signaling vulnerabilities in cancers.

Most cancer cells are dependent on a network of deregulated signaling pathways for survival and are insensitive, or rapidly evolve resistance, to selective inhibitors aimed at a single target. For these reasons, drugs that target more than one protein (polypharmacology) can be clinically advantageous. The discovery of useful polypharmacology remains serendipitous and is challenging to characterize and validate. In this study, we developed a non-genetic strategy for the identification of pathways that drive cancer cell proliferation and represent exploitable signaling vulnerabilities. Our approach is based on using a multitargeted kinase inhibitor, SM1-71, as a tool compound to identify combinations of targets whose simultaneous inhibition elicits a potent cytotoxic effect. As a proof of concept, we applied this approach to a KRAS-dependent non-small cell lung cancer (NSCLC) cell line, H23-KRASG12C Using a combination of phenotypic screens, signaling analyses, and kinase inhibitors, we found that dual inhibition of MEK1/2 and insulin-like growth factor 1 receptor (IGF1R)/insulin receptor (INSR) is critical for blocking proliferation in cells. Our work supports the value of multitargeted tool compounds with well-validated polypharmacology and target space as tools to discover kinase dependences in cancer. We propose that the strategy described here is complementary to existing genetics-based approaches, generalizable to other systems, and enabling for future mechanistic and translational studies of polypharmacology in the context of signaling vulnerabilities in cancers.

Specific covalent inhibition of MALT1 paracaspase suppresses B cell lymphoma growth.

The paracaspase MALT1 plays an essential role in activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) downstream of B cell and TLR pathway genes mutated in these tumors. Although MALT1 is considered a compelling therapeutic target, the development of tractable and specific MALT1 protease inhibitors has thus far been elusive. Here, we developed a target engagement assay that provides a quantitative readout for specific MALT1-inhibitory effects in living cells. This enabled a structure-guided medicinal chemistry effort culminating in the discovery of pharmacologically tractable, irreversible substrate-mimetic compounds that bind the MALT1 active site. We confirmed that MALT1 targeting with compound 3 is effective at suppressing ABC DLBCL cells in vitro and in vivo. We show that a reduction in serum IL-10 levels exquisitely correlates with the drug pharmacokinetics and degree of MALT1 inhibition in vitro and in vivo and could constitute a useful pharmacodynamic biomarker to evaluate these compounds in clinical trials. Compound 3 revealed insights into the biology of MALT1 in ABC DLBCL, such as the role of MALT1 in driving JAK/STAT signaling and suppressing the type I IFN response and MHC class II expression, suggesting that MALT1 inhibition could prime lymphomas for immune recognition by cytotoxic immune cells.

Studies of TAK1-centered polypharmacology with novel covalent TAK1 inhibitors.

Targeted polypharmacology provides an efficient method of treating diseases such as cancer with complex, multigenic causes provided that compounds with advantageous activity profiles can be discovered. Novel covalent TAK1 inhibitors were validated in cellular contexts for their ability to inhibit the TAK1 kinase and for their polypharmacology. Several inhibitors phenocopied reported TAK1 inhibitor 5Z-7-oxozaenol with comparable efficacy and complementary kinase selectivity profiles. Compound 5 exhibited the greatest potency in RAS-mutated and wild-type RAS cell lines from various cancer types. A biotinylated derivative of 5, 27, was used to verify TAK1 binding in cells. The newly described inhibitors constitute useful tools for further development of multi-targeting TAK1-centered inhibitors for cancer and other diseases.

Structure-guided development of covalent TAK1 inhibitors.

TAK1 (transforming growth factor-ß-activated kinase 1) is an essential intracellular mediator of cytokine and growth factor signaling and a potential therapeutic target for the treatment of immune diseases and cancer. Herein we report development of a series of 2,4-disubstituted pyrimidine covalent TAK1 inhibitors that target Cys174, a residue immediately adjacent to the 'DFG-motif' of the kinase activation loop. Co-crystal structures of TAK1 with candidate compounds enabled iterative rounds of structure-based design and biological testing to arrive at optimized compounds. Lead compounds such as 2 and 10 showed greater than 10-fold biochemical selectivity for TAK1 over the closely related kinases MEK1 and ERK1 which possess an equivalently positioned cysteine residue. These compounds are smaller, more easily synthesized, and exhibit a different spectrum of kinase selectivity relative to previously reported macrocyclic natural product TAK1 inhibitors such as 5Z-7-oxozeanol.

Radiological and pathological analysis of LDCT screen detected and surgically resected sub-centimetre lung nodules in 44 asymptomatic patients.

PURPOSE: Once lung cancer is detected due to clinical symptoms or by being visible on chest X-ray, it is usually high stage and non-operable. In order to improve mortality rates in lung cancer, low-dose CT (LDCT) screening of "high risk" individuals is gaining popularity. However, the rate of malignancy in LDCT detected sub-centimetre lung nodules is not clear. We aimed to analyze surgically resected specimens in this patient group to explore cost effectiveness and recommendations for clinical management of these nodules. MATERIAL & METHODS: Our hospital pathology database was searched for sub-centimeter lung nodules detected by LDCT screening which were resected. The patient demographics were collected and the radiologic and pathologic characteristics of those nodules were analyzed. RESULTS: From the records, 44 patients with 46 resected subcentimetre nodules were identified. Patients were selected for surgery based on an irregular shape, growth in size during follow up, family history of lung cancer or personal history of cancer of other sites, previous lung disease, smoking and personal anxiety. Of the 44 patients, 33 were women and the ages ranged from 43 to 76 years (56.75 ± 8.44). All nodules were equal to, or less than 10 mm with a mean diameter of 7.81 ± 1.80 mm (SD). Out of 46 nodules, the pathological diagnoses were: invasive adenocarcinoma (ACa) in 4 (8.7%); adenocarcinoma in situ (AIS) or atypical adenomatous hyperplasia (AAH) in 29 (63%); benign fibrosis/fibrotic scar with inflammation or calcification in 12 (26.1%); an intrapulmonary benign lymph node in 1 (2.2%). Of the ACa, AIS and AAH groups (a total of 31 patients), 77% were women (24 vs. 7). The cancer or pre-cancer nodules (ACa, AIS and AAH) tended to be larger than benign fibrotic scars (P = 0.039). Amongst all characteristics, significant statistical differences were found when the following radiological features were considered: reconstructed nodule shape (P = 0.011), margin (P = 0.003) and ground glass pattern (P = 0.000). The patient's age, the axial morphology of the lesion, relationship to major vessels or visceral pleura and location within the lung parenchyma were not predictive of the pathologic diagnosis. Only one of the 31 patients with a cancer or pre-cancer nodule was a smoker. CONCLUSION: ACa, AIS and AAH nodules detected on LDCT included more women (77%) than men in our cohort. Smoking as inclusive criteria for LDCT screening of lung cancer needs to be further evaluated in the Chinese population. The reconstructed nodule shape, density and margin may help radiologists to identify small cancer and pre-cancer nodules from benign conditions.

Systemic study on the biogenic pathways of yezo'otogirins: total synthesis and antitumor activities of (±)-yezo'otogirin C and its structural analogues.

A systematic study of the biomimetic pathways to yezo'otogirin C under aerobic and anaerobic conditions has been investigated, and both are found to be feasible pathways to the natural product depending on the physiological conditions. Because of the lower activation energy, the aerobic process would be more favorable when the in vivo oxygen level is high. In the course of this study, a highly efficient synthetic route to (±)-yezo'otogirin C has been established in four steps (31% overall yield) from a readily available compound without using any protecting groups. The natural product and its structural analogues exhibited antitumor activities against several human cancer cell lines and appeared to arrest cell cycles in different phases.