Highly Potent and Intestine Specific P-Glycoprotein Inhibitor to Enable Oral Delivery of Taxol.

Taxol is widely used in cancer chemotherapy; however, the oral absorption of Taxol remains a formidable challenge. Since the intestinal p-glycoprotein (P-gp) mediated drug efflux is one of the primary causes, the development of P-gp inhibitor is emerging as a promising strategy to realize Taxol's oral delivery. Because P-gp exists in many tissues, the non-selective P-gp inhibitors would lead to toxicity. Correspondingly, a potent and intestine specific P-gp inhibitor would be an ideal solution to boost the oral absorption of Taxol and avoid exogenous toxicity. Herein, we would like to report a highly potent and intestine specific P-gp inhibitor to enable oral delivery of Taxol in high efficiency. Through a multicomponent reaction and post-modification, various benzofuran-fused-piperidine derivatives were achieved and the biological evaluation identified 16c with potent P-gp inhibitory activity. Notably, 16c was intestine specific and showed almost none absorption (F = 0.82%), but possessing higher efficacy than Encequidar to improve the oral absorption of Taxol. In MDA-MB-231 xenograft model, the oral administration of Taxol and 16c showed high therapeutic efficiency and low toxicity, thus providing a valuable chemotherapy strategy.

Structure-based virtual screening of novel USP5 inhibitors targeting the zinc finger ubiquitin-binding domain.

The equilibrium of cellular protein levels is pivotal for maintaining normal physiological functions. USP5 belongs to the deubiquitination enzyme (DUBs) family, controlling protein degradation and preserving cellular protein homeostasis. Aberrant expression of USP5 is implicated in a variety of diseases, including cancer, neurodegenerative diseases, and inflammatory diseases. In this paper, a multi-level virtual screening (VS) approach was employed to target the zinc finger ubiquitin-binding domain (ZnF-UBD) of USP5, leading to the identification of a highly promising candidate compound 0456-0049. Molecular dynamics (MD) simulations were then employed to assess the stability of complex binding and predict hotspot residues in interactions. The results indicated that the candidate stably binds to the ZnF-UBD of USP5 through crucial interactions with residues ARG221, TRP209, GLY220, ASN207, TYR261, TYR259, and MET266. Binding free energy calculations, along with umbrella sampling (US) simulations, underscored a superior binding affinity of the candidate relative to known inhibitors. Moreover, US simulations revealed conformational changes of USP5 during ligand dissociation. These insights provide a valuable foundation for the development of novel inhibitors targeting USP5.

Discovery of Novel Inhibitors of BRD4 for Treating Prostate Cancer: A Comprehensive Case Study for Considering Water Networks in Virtual Screening and Drug Design.

Androgen receptor (AR) is the primary target for treating prostate cancer (PCa), which inevitably progresses due to drug-resistant mutations. Bromodomain-containing protein 4 (BRD4) has been a new potential drug target for PCa treatment. Herein, we report the rational design and discovery of novel BRD4 inhibitors through computer-aided drug design (CADD), and a hit compound SQ-1 (IC50 = 676 nM) was identified by structure-based virtual screening (SBVS) with the conserved water network. To optimize the structure of SQ-1, the free energy landscape was constructed, and the binding mechanism was explored by characterizing the water profile and the dissociation mechanism. Finally, the compound SQ-17 with improved inhibitory activity (IC50 < 100 nM) was discovered, which showed potent antiproliferative activity against LNCaP. These data highlighted a successful attempt to identify and optimize a small molecule by comprehensive CADD application and provided essential clues for developing novel therapeutics for PCa treatment.

Dissecting the role of ALK double mutations in drug resistance to lorlatinib with in-depth theoretical modeling and analysis.

Anaplastic lymphoma kinase (ALK) is implicated in the genesis of multiple malignant tumors. Lorlatinib stands out as the most advanced and effective inhibitor currently used in the clinic for the treatment of ALK-positive non-small cell lung cancer. However, resistance to lorlatinib has inevitably manifested over time, with double/triple mutations of G1202, L1196, L1198, C1156 and I1171 frequently observed in clinical practice, and tumors regrow within a short time after treatment with lorlatinib. Therefore, elucidating the mechanism of resistance to lorlatinib is paramount in paving the way for innovative therapeutic strategies and the development of next-generation drugs. In this study, we leveraged multiple computational methodologies to delve into the resistance mechanisms of three specific double mutations of ALKG1202R/L1196M, ALKG1202R/L1198F and ALKI1171N/L1198F to lorlatinib. We analyzed these mechanisms through qualitative (PCA, DCCM) and quantitative (MM/GBSA, US) kinetic analyses. The qualitative analysis shows that these mutations exert minimal perturbations on the conformational dynamics of the structural domains of ALK. The energetic and structural assessments show that the van der Waals interactions, formed by the conserved residue Leu1256 within the ATP-binding site and the residues Glu1197 and Met1199 in the hinge domain with lorlatinib, play integral roles in the occurrence of drug resistance. Furthermore, the US simulation results elucidate that the pathways through which lorlatinib dissociates vary across mutant systems, and the distinct environments during the dissociation process culminate in diverse resistance mechanisms. Collectively, these insights provide important clues for the design of novel inhibitors to combat resistance.

A small molecule inhibitor of the UBE2F-CRL5 axis induces apoptosis and radiosensitization in lung cancer.

Protein neddylation is catalyzed by a neddylation activating enzyme (NAE, E1), an E2 conjugating enzyme, and an E3 ligase. In various types of human cancers, the neddylation pathway is abnormally activated. Our previous study validated that the neddylation E2 UBE2F is a promising therapeutic target in lung cancer. Although the NAE inhibitor MLN4924/pevonedistat is currently under clinical investigation as an anti-cancer agent, there are no small molecules available that selectively target UBE2F. Here, we report, for the first time, the discovery, via structure-based virtual screen and chemical optimization, of such a small molecule, designated as HA-9104. HA-9104 binds to UBE2F, reduces its protein levels, and consequently inhibits cullin-5 neddylation. Blockage of cullin-5 neddylation inactivates cullin-RING ligase-5 (CRL5) activity, leading to accumulation of the CRL5 substrate, NOXA, to induce apoptosis. Moreover, HA-9104 appears to form the DNA adduct via its 7-azaindole group to induce DNA damage and G2/M arrest. Biologically, HA-9104 effectively suppresses the growth and survival of lung cancer cells and confers radiosensitization in both in vitro cell culture and in vivo xenograft tumor models. In summary, we discovered a small molecule, designated HA-9104, that targets the UBE2F-CRL5 axis with anti-cancer activity alone or in combination with radiation.

Proteome-Wide Profiling of the Covalent-Druggable Cysteines with a Structure-Based Deep Graph Learning Network.

Covalent ligands have attracted increasing attention due to their unique advantages, such as long residence time, high selectivity, and strong binding affinity. They also show promise for targets where previous efforts to identify noncovalent small molecule inhibitors have failed. However, our limited knowledge of covalent binding sites has hindered the discovery of novel ligands. Therefore, developing in silico methods to identify covalent binding sites is highly desirable. Here, we propose DeepCoSI, the first structure-based deep graph learning model to identify ligandable covalent sites in the protein. By integrating the characterization of the binding pocket and the interactions between each cysteine and the surrounding environment, DeepCoSI achieves state-of-the-art predictive performances. The validation on two external test sets which mimic the real application scenarios shows that DeepCoSI has strong ability to distinguish ligandable sites from the others. Finally, we profiled the entire set of protein structures in the RCSB Protein Data Bank (PDB) with DeepCoSI to evaluate the ligandability of each cysteine for covalent ligand design, and made the predicted data publicly available on website.

p16INK4A-deficiency predicts response to combined HER2 and CDK4/6 inhibition in HER2+ breast cancer brain metastases.

Approximately 50% of patients with metastatic HER2-positive (HER2+) breast cancer develop brain metastases (BCBMs). We report that the tumor suppressor p16INK4A is deficient in the majority of HER2+ BCBMs. p16INK4A-deficiency as measured by protein immunohistochemistry predicted response to combined tucatinib and abemaciclib in orthotopic patient-derived xenografts (PDXs) of HER2 + BCBMs. Our findings establish the rationale for a biomarker-driven clinical trial of combined CDK4/6- and HER2-targeted agents for patients with HER2 + BCBM.

High-throughput glycolytic inhibitor discovery targeting glioblastoma by graphite dots-assisted LDI mass spectrometry.

Malignant tumors will become vulnerable if their uncontrolled biosynthesis and energy consumption engaged in metabolic reprogramming can be cut off. Here, we report finding a glycolytic inhibitor targeting glioblastoma with graphite dots-assisted laser desorption/ionization mass spectrometry as an integrated drug screening and pharmacokinetic platform (GLMSD). We have performed high-throughput virtual screening to narrow an initial library of 240,000 compounds down to the docking of 40 compounds and identified five previously unknown chemical scaffolds as promising hexokinase-2 inhibitors. The best inhibitor (Compd 27) can regulate the reprogrammed metabolic pathway in U87 glioma cells (median inhibitory concentration ~ 11.3 µM) for tumor suppression. Highly effective therapy against glioblastoma has been demonstrated in both subcutaneous and orthotopic brain tumors by synergizing Compd 27 and temozolomide. Our glycolytic inhibitor discovery can inspire personalized medicine targeting reprogrammed metabolisms of malignant tumors. GLMSD enables large, high-quality data for next-generation artificial intelligence-aided drug development.

Discovery of a small molecule inhibitor of cullin neddylation that triggers ER stress to induce autophagy.

Protein neddylation is catalyzed by a three-enzyme cascade, namely an E1 NEDD8-activating enzyme (NAE), one of two E2 NEDD8 conjugation enzymes and one of several E3 NEDD8 ligases. The physiological substrates of neddylation are the family members of cullin, the scaffold component of cullin RING ligases (CRLs). Currently, a potent E1 inhibitor, MLN4924, also known as pevonedistat, is in several clinical trials for anti-cancer therapy. Here we report the discovery, through virtual screening and structural modifications, of a small molecule compound HA-1141 that directly binds to NAE in both in vitro and in vivo assays and effectively inhibits neddylation of cullins 1-5. Surprisingly, unlike MLN4924, HA-1141 also triggers non-canonical endoplasmic reticulum (ER) stress and PKR-mediated terminal integrated stress response (ISR) to activate ATF4 at an early stage, and to inhibit protein synthesis and mTORC1 activity at a later stage, eventually leading to autophagy induction. Biologically, HA-1141 suppresses growth and survival of cultured lung cancer cells and tumor growth in in vivo xenograft lung cancer models at a well-tolerated dose. Taken together, our study has identified a small molecule compound with the dual activities of blocking neddylation and triggering ER stress, leading to growth suppression of cancer cells.

Gossypol inhibits cullin neddylation by targeting SAG-CUL5 and RBX1-CUL1 complexes.

Cullin-RING E3 ligase (CRL) is the largest family of E3 ubiquitin ligase, responsible for ubiquitylation of ∼20% of cellular proteins. CRL plays an important role in many biological processes, particularly in cancers due to abnormal activation. CRL activation requires neddylation, an enzymatic cascade transferring small ubiquitin-like protein NEDD8 to a conserved lysine residue on cullin proteins. Recent studies have validated that neddylation is an attractive anticancer target. In this study, we report the establishment of an Alpha-Screen-based high throughput screen (HTS) assay for in vitro CUL5 neddylation, and screened a library of 17,000 compounds including FDA approved drugs, natural products and synthetic drug-like small-molecule compounds. Gossypol, a natural compound derived from cotton seed, was identified as an inhibitor of cullin neddylation. Biochemical studies showed that gossypol blocked neddylation of both CUL5 and CUL1 through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and CUL5-H572 plays a key role for gossypol binding. On cellular level, gossypol inhibited cullin neddylation in a variety of cancer cell lines and selectively caused accumulation of NOXA and MCL1, the substrates of CUL5 and CUL1, respectively, in multiple cancer cell lines. Combination of gossypol with specific MCL1 inhibitor synergistically suppress growth of human cancer cells. Our study revealed a previously unknown anti-cancer mechanism of gossypol with potential to develop a new class of neddylation inhibitors.

Drug Discovery Targeting Anaplastic Lymphoma Kinase (ALK).

As a receptor tyrosine kinase of insulin receptor (IR) subfamily, anaplastic lymphoma kinase (ALK) has been validated to play important roles in various cancers, especially anaplastic large cell lymphoma (ALCL), nonsmall cell lung cancer (NSCLC), and neuroblastomas. Currently, five small-molecule inhibitors of ALK, including Crizotinib, Ceritinib, Alectinib, Brigatinib, and Lorlatinib, have been approved by the U.S. Food and Drug Administration (FDA) against ALK-positive NSCLCs. Novel type-I1/2 and type-II ALK inhibitors with improved kinase selectivity and enhanced capability to combat drug resistance have also been reported. Moreover, the "proteolysis targeting chimera" (PROTAC) technique has been successfully applied in developing ALK degraders, which opened a new avenue for targeted ALK therapies. This review provides an overview of the physiological and biological functions of ALK, the discovery and development of drugs targeting ALK by focusing on their chemotypes, activity, selectivity, and resistance as well as potential therapeutic strategies to overcome drug resistance.

Discovery of 3,6-diaryl-1H-pyrazolo[3,4-b]pyridines as potent anaplastic lymphoma kinase (ALK) inhibitors.

A new series of 3,6-diaryl-1H-pyrazolo[3,4-b]pyridine compounds have been discovered as potent anaplastic lymphoma kinase (ALK) inhibitors. The 4-hydroxyphenyl in the 6-position of 1H-pyrazolo[3,4-b]pyridine were crucial and a fluorine atom substitution could give promising inhibitory activity. The IC50 of compound 9v against ALK was up to 1.58 nM and a binding mechanism was proposed.

Improving orthotopic mouse models of patient-derived breast cancer brain metastases by a modified intracarotid injection method.

Breast cancer brain metastasis (BCBM) remains a major clinical problem. Approximately 10-16% of patients with breast cancer develop brain metastases (BCBM). However, no systemic therapy has gained regulatory approval for the specific treatment of BCBM and this remains an area of persistent, unmet medical need. Rapid, predictive and clinically-relevant animal models are critical to study the biology of brain metastases and to identify effective therapeutic approaches for patients with BCBM. Here, we describe a method for efficient establishment of orthotopic mouse models of patient-derived brain metastases via an improved intracarotid injection protocol that permits tumor cell growth in the unique brain microenvironment without compromising the blood-brain barrier (BBB). We demonstrate that our newly improved models of patient-derived brain metastases recapitulate the histologic, molecular, and genetic characteristics of their matched patient tumor specimens and thus represent a potentially powerful tool for pre-clinical and translational research.

Structure-Based Drug Design and Identification of H2O-Soluble and Low Toxic Hexacyclic Camptothecin Derivatives with Improved Efficacy in Cancer and Lethal Inflammation Models in Vivo.

Camptothecin (CPT) has been shown to block disassembly of the topoisomerase I (Topo I)/DNA cleavable complex. However, the poor aqueous solubility, intrinsic instability, and severe toxicity of CPTs have limited their clinical applications. Herein, we report the design and synthesis of H2O-soluble and orally bioavailable hexacyclic CPT derivatives. By analysis of a virtual chemical library and cytotoxicity screening in vitro, 9 and 11 were identified as potential prodrugs and chosen for further characterization in vivo. Both compounds exhibited remarkable anticancer and anti-inflammation efficacies in animals and improved drug-like profiles.

Importance of protein flexibility in molecular recognition: a case study on Type-I1/2 inhibitors of ALK.

Anaplastic lymphoma kinase (ALK) has been regarded as a promising target for the therapy of various cancers. A large number of ALK inhibitors with diverse scaffolds have been discovered, and most of them belong to Type-I inhibitors that only occupy the ATP-binding pocket. Recently, we reported a series of novel and potent Type-I1/2 inhibitors of ALK with the 1-purine-3-piperidinecarboxamide scaffold, which can bind to both the ATP-binding site of ALK and the adjacent hydrophobic allosteric pocket. In this study, the binding mechanisms of these Type-I1/2 ALK inhibitors were elucidated by multiple molecular modeling techniques. The calculation results demonstrate that the ensemble docking based on multiple protein structures and the MM/PB(GB)SA calculations based on molecular dynamics (MD) simulations yield better predictions than conventional rigid receptor docking (Glide, Surflex-Dock, and Autodock Vina), highlighting the importance of incorporating receptor flexibility in the predictions of binding poses and binding affinities of Type-I1/2 ALK inhibitors. Furthermore, the umbrella sampling (US) simulations and MM/GBSA binding free energy decomposition analyses indicate that Leu1122, Leu1198, Gly1202 and Glu1210 in the hinge region and Glu1197, Ile1171, Phe1174, Ile1179, His1247, Ile1268, Asp1270 and Phe1271 in the allosteric pocket of ALK are the key residues for determining the relative binding strength of the studied inhibitors. Besides, we found that the most potent inhibitor (001-017) tends to form stronger transient interactions with residues along the dissociation channel due to the high electronegativity of its bulky 4-(trifluoromethoxy) phenylamine tail. As a whole, both the stronger binding affinity and the higher energetic barrier (which may prolong the drug-target residence time) of 001-017 contribute to its excellent anti-proliferation activity against ALK-positive cancer cells.

Combating Drug-Resistant Mutants of Anaplastic Lymphoma Kinase with Potent and Selective Type-I1/2 Inhibitors by Stabilizing Unique DFG-Shifted Loop Conformation.

Targeted inhibition of anaplastic lymphoma kinase (ALK) dramatically improved therapeutic outcomes in the treatment of ALK-positive cancers, but unfortunately patients invariably progressed due to acquired resistance mutations in ALK. Currently available drugs are all type-I inhibitors bound to the ATP-binding pocket and are most likely to be resistant in patients harboring genetic mutations surrounding the ATP pocket. To overcome drug resistance, we rationally designed a novel kind of "bridge" inhibitor, which specially bind into an extended hydrophobic back pocket adjacent to the ATP-binding site of ALK. The novel type-I1/2 inhibitors display excellent antiproliferation activity against ALK-positive cancer cells and appear superior to two clinically used drugs, crizotinib and ceritinib. Structural and molecular modeling analyses indicate that the inhibitor induces dramatic conformational transition and stabilizes unique DFG-shifted loop conformation, enabling persistent sensitivity to different genetic mutations in ALK. These data highlight a rationale for further development of next-generation ALK inhibitors to combat drug resistance.

How Does the L884P Mutation Confer Resistance to Type-II Inhibitors of JAK2 Kinase: A Comprehensive Molecular Modeling Study.

Janus kinase 2 (JAK2) has been regarded as an essential target for the treatment of myeloproliferative neoplasms (MPNs). BBT594 and CHZ868, Type-II inhibitors of JAK2, illustrate satisfactory efficacy in preclinical MPNs and acute lymphoblastic leukemia (ALL) models. However, the L884P mutation of JAK2 abrogates the suppressive effects of BBT594 and CHZ868. In this study, conventional molecular dynamics (MD) simulations, umbrella sampling (US) simulations and MM/GBSA free energy calculations were employed to explore how the L884P mutation affects the binding of BBT594 and CHZ868 to JAK2 and uncover the resistance mechanism induced by the L884P mutation. The results provided by the US and MD simulations illustrate that the L884P mutation enhances the flexibility of the allosteric pocket and alters their conformations, which amplify the conformational entropy change (-TΔS) and weaken the interactions between the inhibitors and target. Additionally, the structural analyses of BBT594 and CHZ868 in complex with the WT JAK2 illustrate that the drug tail with strong electronegativity and small size located in the allosteric pocket of JAK2 may enhance anti-resistance capability. In summary, our results highlight that both of the changes of the conformational entropies and enthalpies contribute to the L884P-induced resistance in the binding of two Type-II inhibitors into JAK2 kinase.

In Silico Exploration for Novel Type-I Inhibitors of Tie-2/TEK: The Performance of Different Selection Strategy in Selecting Virtual Screening Candidates.

The receptor tyrosine kinase Tie-2 is involved in vessel remodeling and maturation, and has been regarded as a potential target for the treatment of various solid tumors. The absence of novel, potent and selective inhibitors severely hampers the understanding of the therapeutic potential of Tie-2. In the present work, we describe the discovery of novel type-I inhibitors of Tie-2 by structure-based virtual screening. Preliminary SAR was also performed based on one active compound, and several novel inhibitors with low micro-molar affinity were discovered. To directly compare the efficiency between different filtering strategies in selecting VS candidates, two methods were separately carried out to screen the same chemical library, and the selected VS candidates were then experimentally assessed by in vitro enzymatic assays. The results demonstrate that the hit rate is improved when stricter drug-likeness criteria and less number of molecules for clustering analysis are used, and meanwhile, the molecular diversity of the compounds still maintains. As a case study of TIE-2, the information presented in this work underscores the importance of selecting an appropriate selection strategy in VS campaign, and the novel inhibitors identified and the detailed binding modes of action provide a starting point for further hit-to-lead optimization process.

DNA-PKcs, a novel functional target of acriflavine, mediates acriflavine's p53-dependent synergistic anti-tumor efficiency with melphalan.

Acriflavine (ACF), a known antibacterial drug, has recently been recognized as a suitable candidate for cancer chemotherapy. However, the molecular target of ACF is not fully understood, which limits its application in cancer therapy. In this study, we established a structure-specific probe-based pull-down approach to comprehensively profile the potential target of ACF, and we identified DNA dependent protein kinase catalytic subunit (DNA-PKcs) as the direct target of ACF. Since DNA-PKcs facilitates the repair process following DNA double-strand breaks, we further developed a drug combination strategy that combined ACF with the bifunctional alkylating agent melphalan, which exerted a p53-dependent synergistic efficacy against human cancer cells both in vitro and in vivo. With these findings, our study demonstrated that structure-specific probe-based pull-down approaches can be used to identify new functional target of drug, and provided novel opportunities for the development of ACF-based antitumor chemotherapies.

Discovery of a novel ROCK2 inhibitor with anti-migration effects via docking and high-content drug screening.

Rho-associated protein kinase (ROCK) mediated the reorganization of the actin cytoskeleton and has been implicated in the spread and metastatic process of cancer. In this study, structure-based high-throughput virtual screening was used to identify candidate compounds targeting ROCK2 from a chemical library. Moreover, high-content screening based on neurite outgrowth of SH-SY5Y cells (a human neuroblastoma cell line) was used for accelerating the identification of compounds with characteristics of ROCK2 inhibitors. The effects of bioactive ROCK2 inhibitor candidates were further validated using other bioassays including cell migration and wound healing in SH-SY5Y cells. Through the combined virtual and high-content drug screening, the compound 1,3-benzodioxol-5-yl[1-(5-isoquinolinylmethyl)-3-piperidinyl]-methanone (BIPM) was identified as a novel and potent ROCK2 inhibitor. Exposure of SH-SY5Y cells to BIPM led to significant changes in neurite length, cell migration and actin stress fibers. Further experiments demonstrated that BIPM was able to significantly inhibit phosphorylation of cofilin, a regulatory protein of actin cytoskeleton. These results suggest that BIPM could be considered as a promising scaffold for the further development of ROCK2 inhibitors for anti-cancer metastasis.