Target For Anticancer Drug Discovery Research Articles

A novel peptide CP29L, selected from the phage displayed cyclic random heptapeptide library, demonstrates its potent inhibitory effects to liver cancer HCCLM3 cells by targeting FOXM1.

FOXM1 is the "Achilles' heel" of cancers and hence the potential therapeutic target for anticancer drug discovery. In this work, we selected high affinity peptides against the protein of human DNA binding domain of FOXM1 (FOXM1-DBD) from the disulfide-constrained, phage displayed random cyclic heptapeptide library Ph.D.-C7C. We obtained a novel peptide, 9R-CP29L, which was identified to be a potent anticancer peptide with IC50 values of 9.0 and 11.1μMat 24h for HCCLM3 and MD-MBA-231cells respectively. Molecular docking, CETSA, ITC and immunoblot assays demonstrated that 9R-CP29L can potentially specifically bind to FOXM1-DBD with a Kd value of 1.25μM and reduced the expression of FOXM1. In addition, Annexin V/PI flow cytometry, AO/EB staining, PI flow cytometry, clone formation and Transwell assays revealed that 9R-CP29L also induced cell apoptosis and cell cycle arrest while inhibited the proliferation and migration of HCCLM3 cells. The findings were further supported by the results of qRT-PCR and immunoblot assays for the associated gene (CMYC, CDC25B, BAX, CASPASE3 and MMP2, etc) expression in HCCLM3 cells. Finally, in vivo experiment showed that 9R-CP29 significantly reduced the tumor growth and downregulated the expression of FOXM1 in HCCLM3 xenograft nude mouse models. Taking together, our work provides a novel FOXM1 targeted peptide which has potential in both anticancer drug development and scientific researches.

Design and synthesis of (E)-3-benzylideneindolin-2-one derivatives as potential allosteric inhibitors of Aurora A kinase.

The mitotic kinase Aurora A, a pivotal regulator of the cell cycle, is overexpressed in various cancers and has emerged as one of the most promising targets for anticancer drug discovery. However, the lack of specificity and potential toxicity have impeded clinical trials involving orthosteric inhibitors. In this study, allosteric sites of Aurora A were predicted using the AlloReverse web server. Based on the non-ATP competitive inhibitor Tripolin A and molecular docking information targeting the desired allosteric site 3 of Aurora A, a series of (E)-3-benzylideneindolin-2-one derivatives were designed and synthesized. Compared to Tripolin A, our compounds AK09, AK34 and AK35 have stronger inhibitory effects and can be further investigated as potential allosteric inhibitors. Moreover, the compound AK34 with the strongest inhibitory activity (IC50 = 1.68 μM) has a high affinity for Aurora A (K D = 216 nM). According to the analysis of the structure-activity relationship of the compounds and the results of their molecular docking models, these compounds tend to act on the allosteric site 3 of Aurora A.

Cheminformatics-aided discovery of potential allosteric site modulators of ubiquitin-specific protease 7

Ubiquitin-specific peptidase 7 (USP7) is a deubiquitinating enzyme that mediates the stability and activity of numerous proteins. At basal expression levels, USP7 stabilizes p53 protein, even in the presence of excess MDM2. However, its overexpression leads to the deubiquitination of MDM2 at a rate faster than p53, leading to p53 degradation and pro-tumorigenic roles. Consequently, it is an attractive target for anticancer drug discovery via the modulation of its allosteric site from which the protein is activated. In this study, molecular modeling techniques and cheminformatics approaches were employed to unravel the potential of eighty compounds to serve as its allosteric site modulators. The compounds were initially subjected to virtual screening. Subsequently, the binding free energies of the top four compounds with the highest binding affinities were calculated, and their drug-likeness, and pharmacokinetic and toxicity profiles were evaluated. Ultimately, the complexes of the protein and hit compounds were subjected to a 100 nanoseconds (ns) molecular dynamics simulation. The results of the study revealed eight compounds from the compound library with docking scores ranging from − 7.491 to -11.43 kcal/mol, compared to P217564, which exhibited a docking score of -5.671 kcal/mol. The top four compounds with the highest affinities possessed drug-like properties, and good pharmacokinetic and toxicity profiles, and their predicted inhibitory potentials showed they will be effective at minimal concentration. Also, molecular dynamics simulation confirmed the stability of the protein-ligand complexes. Conclusively, the compounds identified in this study are worthy of further evaluation for the development of allosteric site modulators of USP7.

Structure-Based De Novo Design for the Discovery of Miniprotein Inhibitors Targeting Oncogenic Mutant BRAF.

Being a component of the Ras/Raf/MEK/ERK signaling pathway crucial for cellular responses, the VRAF murine sarcoma viral oncogene homologue B1 (BRAF) kinase has emerged as a promising target for anticancer drug discovery due to oncogenic mutations that lead to pathway hyperactivation. Despite the discovery of several small-molecule BRAF kinase inhibitors targeting oncogenic mutants, their clinical utility has been limited by challenges such as off-target effects and suboptimal pharmacological properties. This study focuses on identifying miniprotein inhibitors for the oncogenic V600E mutant BRAF, leveraging their potential as versatile drug candidates. Using a structure-based de novo design approach based on binding affinity to V600E mutant BRAF and hydration energy, 39 candidate miniprotein inhibitors comprising three helices and 69 amino acids were generated from the substructure of the endogenous ligand protein (14-3-3). Through in vitro binding and kinase inhibition assays, two miniproteins (63 and 76) were discovered as novel inhibitors of V600E mutant BRAF with low-micromolar activity, with miniprotein 76 demonstrating a specific impediment to MEK1 phosphorylation in mammalian cells. These findings highlight miniprotein 76 as a potential lead compound for developing new cancer therapeutics, and the structural features contributing to its biochemical potency against V600E mutant BRAF are discussed in detail.

EGFR degraders in non-small-cell lung cancer: Breakthrough and unresolved issue.

The epidermal growth factor receptor (EGFR) has been well validated as a therapeutic target for anticancer drug discovery. Osimertinib has become the first globally accessible third-generation EGFR inhibitor, representing one of the most advanced developments in non-small-cell lung cancer (NSCLC) therapy. However, a tertiary Cys797 to Ser797 (C797S) point mutation has hampered osimertinib treatment in patients with advanced EGFR-mutated NSCLC. Several classes of fourth-generation EGFR inhibitors were consequently discovered with the aim of overcoming the EGFRC797S mutation-mediated resistance. However, no clinical efficacy data of the fourth-generation EGFR inhibitors were reported to date, and EGFRC797S mutation-mediated resistance remains an "unmet clinical need." Proteolysis-targeting chimeric molecules (PROTACs) obtained from EGFR-TKIs have been developed to target drug resistance EGFR in NSCLC. Some PROTACs are from nature products. These degraders compared with EGFR inhibitors showed better efficiency in their cellular potency, inhibition, and toxicity profiles. In this review, we first introduce the structural properties of EGFR, the resistance, and mutations of EGFR, and then mainly focus on the recent advances of EGFR-targeting degraders along with its advantages and outstanding challenges.

Design, synthesis, and biological evaluation of new biaryl derivatives of cycloalkyl diacetamide bearing chalcone moiety as type II c-MET kinase inhibitors.

Many human cancers have been associated with the deregulation of the mesenchymal-epithelial transition factor tyrosine kinase (MET) receptor, a promising drug target for anticancer drug discovery. Herein, we report the discovery of a novel structure of potent chalcone-based derivatives type II c-Met inhibitors which are comparable to Foretinib (IC50 = 14 nM) as a potent reference drug. Based on our design strategy, we also expected an anti-tubulin activity for the compounds. However, the weak inhibitory effects on microtubules were confirmed by cell cycle analyses implicated that the observed cytotoxicity against HeLa cells probably was not derived from tubulin inhibition. Compounds 14q and 14k with IC50 values of 24 nM and 45 nM, respectively, demonstrated favorable inhibition of MET kinase activity, and desirable bonding interactions in the ligand-MET enzyme complex stability in molecular docking studies.

The Development of HDAC and Tubulin Dual-Targeting Inhibitors for Cancer Therapy.

Histone deacetylases (HDACs) are a class of enzymes that are responsible for the removal of acetyl groups from the ε-N-acetyl lysine of histones, allowing histones to wrap DNA more tightly. HDACs play an essential role in many biological processes, such as gene regulation, transcription, cell proliferation, angiogenesis, migration, differentiation and metastasis, which make it an excellent target for anticancer drug discovery. The search for histone deacetylase inhibitors (HDACis) has been intensified, with numerous HDACis being discovered, and five of them have reached the market. However, currently available HDAC always suffers from several shortcomings, such as limited efficacy, drug resistance, and toxicity. Accordingly, dual-targeting HDACis have attracted much attention from academia to industry, and great advances have been achieved in this area. In this review, we summarize the progress on inhibitors with the capacity to concurrently inhibit tubulin polymerization and HDAC activity and their application in cancer treatment.

Discovery of anticancer compound possessing potential to bind γ-secretase catalytic subunit and inhibit notch promoter activity

Gamma secretase (GS) is an important therapeutic target in anticancer drug discovery. Increased GS activity activates notch signaling pathway which is associated with cancer stemness and drug resistance in cancer cells. A total of 69,075 natural and their derivative compounds were screened to identify the lead compound on the basis of in silico GS catalytic domain binding potential and in vitro selective anticancer efficacy. STOCK1N-23234 showed higher dock score (−11.82) compared to DAPT (−9.2) in molecular docking experiment and formed hydrogen bond with the key amino acid (Asp385) involve in catalysis process. Molecular dynamics (MD) simulation parameters (RMSD, RMSF, Rg, SASA and hydrogen bond formation) revealed that the STOTCK1N-23234 formed structurally and energetically stable complex with the GS catalytic domain with lower binding energy (−22.79 kcal/mol) compared to DAPT (−16.22 kcal/mol). STOCK1N-23234 showed better toxicity (up to 60%) against colon and breast cancer cells (HCT-116 and MDA-MB-453) at 1–70 µM concentration. Interestingly, STOCK1N-23234 did not showed cytotoxicity against human normal breast cells (MCF-10A). STOCK1N-23234 treatment significantly decreased sphere formation, notch promoter activity, and transcription of notch target genes (Hes-1 and Hey-1) in HCT-116 cells derived colonosphere. Confocal microscopy revealed that STOTCK1N-23234 treatment at test concentration induced apoptosis related morphological changes, reduced mitochondria membrane potential and increased reactive oxygen species production in HCT-116 cells compared to non-treated cells. In conclusion, STOCK1N-23234 is a novel lead natural anticancer compound which requires in depth validation in cancer preclinical models. Communicated by Ramaswamy H. Sarma

Design of new small molecules derived from indolin-2-one as potent TRKs inhibitors using a computer-aided drug design approach

Tropomyosin receptor kinase (TRKs) enzymes are responsible for cancers associated with the neurotrophic tyrosine kinase receptor gene fusion and are identified as effective targets for anticancer drug discovery. A series of small-molecule indolin-2-one derivatives showed remarkable biological activity against TRKs enzymatic activity. These small molecules could have an excellent profile for pharmaceutical application in the treatment of cancers caused by TRKs activity. The aim of this study is to modify the structure of these molecules to obtain new molecules with improved TRK inhibitory activity and pharmacokinetic properties favorable to the design of new drugs. Based on these series, we carried out a 3D-QSAR study. As a result, robust and reliable CoMFA and CoMSIA models are developed and applied to the design of 11 new molecules. These new molecules have a biological activity superior to the most active molecule in the starting series. The eleven designed molecules are screened using drug-likeness, ADMET proprieties, molecular docking, and MM-GBSA filters. The results of this screening identified the T1, T3, and T4 molecules as the best candidates for strong inhibition of TRKs enzymatic activity. In addition, molecular dynamics simulations are performed for TRK free and complexed with ligands T1, T3, and T4 to evaluate the stability of ligand-protein complexes over the simulation time. On the other hand, we proposed experimental synthesis routes for these newly designed molecules. Finally, the designed molecules T1, T2, and T3 have great potential to become reliable candidates for the conception of new drug inhibitors of TRKs. Communicated by Ramaswamy H. Sarma

Discovery of a 1,6-naphthyridin-4-one-based AXL inhibitor with improved pharmacokinetics and enhanced in vivo antitumor efficacy

The receptor tyrosine kinase AXL has emerged as an attractive target in anticancer drug discovery. Herein, we described the discovery of a new series of 1,6-naphthyridin-4-one derivatives as potent AXL inhibitors. Starting from a low in vivo potency compound 9 which was previously reported by our group, we utilized structure-based drug design and scaffold hopping strategies to discover potent AXL inhibitors. The privileged compound 13c was a highly potent and orally bioavailable AXL inhibitor with an IC50 value of 3.2 ± 0.3 nM. Compound 13c exhibited significantly improved in vivo antitumor efficacy in AXL-driven tumor xenograft mice, causing tumor regression at well-tolerated dose, and demonstrated favorable pharmacokinetic properties (MRT = 16.5 h, AUC0-∞ = 59,815 ng h/mL) in Sprague-Dawley rats. These results suggest that 13c is a promising therapeutic candidate for AXL-targeting cancer treatment.

Structure-Based Discovery of Potent, Orally Bioavailable Benzoxazepinone-Based WD Repeat Domain 5 Inhibitors.

The chromatin-associated protein WDR5 (WD repeat domain 5) is an essential cofactor for MYC and a conserved regulator of ribosome protein gene transcription. It is also a high-profile target for anti-cancer drug discovery, with proposed utility against both solid and hematological malignancies. We have previously discovered potent dihydroisoquinolinone-based WDR5 WIN-site inhibitors with demonstrated efficacy and safety in animal models. In this study, we sought to optimize the bicyclic core to discover a novel series of WDR5 WIN-site inhibitors with improved potency and physicochemical properties. We identified the 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one core as an alternative scaffold for potent WDR5 inhibitors. Additionally, we used X-ray structural analysis to design partially saturated bicyclic P7 units. These benzoxazepinone-based inhibitors exhibited increased cellular potency and selectivity and favorable physicochemical properties compared to our best-in-class dihydroisoquinolinone-based counterparts. This study opens avenues to discover more advanced WDR5 WIN-site inhibitors and supports their development as novel anti-cancer therapeutics.

Identification of promising inhibitors against breast cancer disease by targeting NUDIX hydrolase 5 (NUDT5) biomolecule

It is well documented that NUDT5 enzyme inhibition in breast cancer cell lines arrest cancer cells growth, invasiveness and migration. The NUDT5 enzyme enhances breast cancer aggressiveness and act as key regulator of oncogenic pathways. Similarly, the NUDT5 enzyme plays a primer role in ATP-dependent cellular processes and proliferation in breast cancer. Thus, the NUDT5 enzyme plays a profound contribution in promoting breast cancers carcinogenesis and could be an ideal target for anti-cancer drug discovery. In this work, LAS_51382001, LAS_51177972 and LAS_51380924 with binding energy of −12.64 kcal/mol, −11.59 kcal/mol and −10.01 kcal/mol, respectively were filtered as lead molecules. The control molecule binding energy was −10.87 kcal/mol. The system dynamics were found uniform in molecular dynamics simulation studies and observed with no major structural changes. Among the leads, the LAS_51177972 showed the most stable binding energy values. The MM-GBSA binding energy of the compound was −37.07 kcal/mol and MM-PBSA binding energy of −43.56 kcal/mol. Similarly, the compound revealed very stable WaterSwap absolute binding energy values; Bennett’s, TI and FEP energy of −36.2 kcal/mol, −36.13 kcal/mol and −36.58 kcal/mol, respectively. Similarly, the leads reported very favorable physicochemical properties, water solubility, pharmacokinetics, druglikeness and medicinal chemistry properties. In a nutshell, the compounds are potent in term of the current computational study however, need to be subjected to experimental studies.Communicated by Ramaswamy H. Sarma

Probing a distinct druggable tubulin binding site with gatorbulins 1–7, their metabolic and physicochemical properties, and pharmacological consequences

Microtubules, consisting of α/β-tubulin heterodimers, are prime targets for anticancer drug discovery. Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2–7 (1b–1g) of this highly modified cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and β-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDR1/P-gp, showing similar permeability with and against transporter gradient, indicating that GB1 (1a) is a poor P-gp substrate.

Discovery of novel resorcinol biphenyl ether-based macrocyclic small molecules as PD-1/PD-L1 inhibitors with favorable pharmacokinetics for cancer immunotherapy

Programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) is one of the most promising immune checkpoints (ICs) in tumor immunology and has been actively pursued as a target for anticancer drug discovery. Based on our previous research in small molecule PD-1/PD-L1 modulators, we designed and synthesized a series of resorcinol biphenyl ether-bearing macrocyclic compounds and evaluated their anti-PD-1/PD-L1 activities. Among them, compound 8d exhibited the highest inhibitory activity against PD-1/PD-L1 interaction with IC50 of 259.7 nM in the homogenous time-resolved fluorescence (HTRF) assay. In addition, 8d displayed in vitro immunomodulatory effects by promoting HepG2 cell death in a HepG2/Jurkat cell co-culture model. Furthermore, 8d effectively inhibited tumor growth (TGI = 74.6% at 40 mg/kg) in a melanoma tumor model in mice without causing obvious toxicity. Moreover, 8d exhibited favorable pharmacokinetics [e.g. high stability, reasonable half-life, and good oral bioavailability (F = 21.5%)]. Finally, molecular modeling studies showed that 8d bound to PD-L1 with high affinity. These results suggest that 8d may serve as a starting point for further development of macrocyclic small molecule-based PD-1/PD-L1 inhibitors for cancer treatment.

Synthetic PARP-1 Inhibitors Reported During the Last Decade

Background: Cancer is the world's second largest cause of death and is responsible for an estimated 9.6 million mortalities in 2018. Poly-ADP-ribose polymerases (PARPs) are enzymes and family of proteins, involved in many cellular processes, including DNA repair, gene regulation, chromatin remodeling, and apoptosis. The first characterized and best known member of the PARP family is poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is a major protein for DNA single-strand breaks in the BER pathway (base excision repair) (SSBs). Objective: The objective of this article was to compile synthetic PARP-1 inhibitors reported in the last decade Methods: In the present manuscript, bibliographic investigation was carried out by scrutinizing peerreviewed articles from online/offline databases. The inclusion criteria consisted of the most relevant studies indicating the relationship between PARP-1 and cancer in textbooks/edited books and peer-reviewed papers from scientific databases, like SCOPUS, PUBMED, NISCAIR, and Google Scholar since 2010 to 2020. Only the studies published in English language were searched/considered. The exclusion criteria consisted of the studies on other PARP isoforms than PARP-1. The studies thus obtained were classified according to the heterocyclic moieties, year of publication, etc. The data compiled in this article is a systematic review of the reported studies. Results: The literature reports indicated that a number of PARP-1 inhibitors reported have IC50 value in nanomolar concentration. Conclusion: PARP-1 is an essential target for anti-cancer drug discovery. Further, research for more effective and safe PARP-1 inhibitors is carried out and we may discover some novel PARP-1 inhibitors in the near future.

Design, synthesis, and anticancer activity of some novel 1H-benzo[d]imidazole-5-carboxamide derivatives as fatty acid synthase inhibitors

Multiple malignancies exhibit aberrant FASN expression, associated with enhanced de novo lipogenesis to meet the metabolic demands of rapidly proliferating tumour cells. Furthermore, elevated FASN expression has been linked to tumour aggressiveness and poor prognosis in a variety of malignant tumours, making FASN is an attractive target for anticancer drug discovery. Herein, we report the de novo design and synthesis of (2-(2-hydroxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperazin-1-yl)methanone derivatives as novel FASN inhibitors with potential therapeutic applications in breast and colorectal cancers. Twelve (2-(2-hydroxyphenyl)-1H-benzo[d]imidazol-5-yl)(piperazin-1-yl)methanone derivatives (CTL) were synthesized and evaluated for FASN inhibition and cytotoxicity against colon cancer (HCT-116, Caco-2 cell lines), breast cancer (MCF-7 cell line) and normal cell line (HEK-293). Compounds CTL-06 and CTL-12 were chosen as the most promising lead molecules based on FASN inhibition and selective cytotoxicity profiles against colon and breast cancer cell lines. Compounds CTL-06 and CTL-12 demonstrate promising FASN inhibitory activity at IC50 of 3 ± 0.25 µM and 2.5 ± 0.25 µM when compared to the FASN inhibitor orlistat, which has an IC50 of 13.5 ± 1.0 µM. Mechanistic investigations on HCT-116 revealed that CTL-06 and CTL-12 treatment led to cell cycle arrest in Sub-G1/S phase along with apoptosis induction. Western blot studies indicated that CTL-06 and CTL-12 inhibited FASN expression in a dose-dependent manner. CTL-06 and CTL-12 treatment of HCT-116 cells enhanced caspase-9 expression in a dose-dependent manner, while upregulating proapoptotic marker Bax and downregulating antiapoptotic Bcl-xL. Molecular docking experiments of CTL-06 and CTL-12 with FASN enzyme revealed the mode of binding of these analogues in the KR domain of the enzyme.

Dihydropyrimidone Derivatives as Thymidine Phosphorylase Inhibitors: Inhibition Kinetics, Cytotoxicity, and Molecular Docking.

Overexpression of the thymidine phosphorylase (TP) enzyme induces angiogenesis, which eventually leads to metastasis and tumor growth. The crucial role of TP in cancer development makes it an important target for anticancer drug discovery. Currently, there is only one US-FDA-approved drug, i.e., Lonsurf, a combination of trifluridine and tipiracil, for the treatment of metastatic colorectal cancer. Unfortunately, numerous adverse effects are associated with its use, such as myelosuppression, anemia, and neutropenia. Since the last few decades, the discovery of new, safe, and effective TP inhibitory agents has been rigorously pursued. In the present study, we evaluated a series of previously synthesized dihydropyrimidone derivatives 1-40 for their TP inhibitory potential. Compounds 1, 12, and 33 showed a good activity with IC50 = 314.0 ± 0.90, 303.5 ± 0.40, and 322.6 ± 1.60 µM, respectively. The results of mechanistic studies revealed that compounds 1, 12, and 33 were the non-competitive inhibitors. These compounds were also evaluated for cytotoxicity against 3T3 (mouse fibroblast) cells and were found to be non-cytotoxic. Finally, the molecular docking suggested the plausible mechanism of non-competitive inhibition of TP. The current study thus identifies some dihydropyrimidone derivatives as potential inhibitors of TP, which can be further optimized as leads for cancer treatment.

Design, synthesis and biological evaluation of novel indolin-2-one derivatives as potent second-generation TRKs inhibitors

Tropomyosin receptor kinases (TRKs) are effective targets for anti-cancer drug discovery. The first-generation type I TRKs inhibitors, larotrectinib and entrectinib, exhibit durable disease control in the clinic. The emergence of acquired resistance mediated by secondary mutations in the TRKs domain significantly reduces the therapeutic efficacy of these two drugs, indicating an unmet clinical need. In this study, we designed a potent and orally bioavailable TRK inhibitor, compound 24b, using a molecular hybridization strategy. Compound 24b exhibited significant inhibitory potency against multiple TRK mutants in both biochemical and cellular assays. Furthermore, compound 24b induced apoptosis of Ba/F3-TRKAG595R and Ba/F3-TRKAG667C cells in a dose-dependent manner. Additionally, compound 24b exhibited moderate kinase selectivity. In vitro stability revealed that compound 24b showed excellent plasma stability (t1/2 > 289.1 min) and moderate liver microsomal stability (t1/2 = 44.3 min). Pharmacokinetic studies have revealed that compound 24b is an orally bioavailable TRK inhibitor with a good oral bioavailability of 116.07%. These results indicate that compound 24b be used as a lead molecule for further modifications to overcome drug-resistant mutants of TRK.

Therapeutic disruption of RAD52-ssDNA complexation via novel drug-like inhibitors.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23-1200 μM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3-8 μM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Structural Optimization and Anticancer Activity of Polo-like Kinase 1 (Plk1) Polo-Box Domain (PBD) Inhibitors and Their Prodrugs.

Polo-like kinase 1 (Plk1), a mitotic kinase whose activity is widely upregulated in various human cancers, is considered an attractive target for anticancer drug discovery. Aside from the kinase domain, the C-terminal noncatalytic polo-box domain (PBD), which mediates the interaction with the enzyme's binding targets or substrates, has emerged as an alternative target for developing a new class of inhibitors. Various reported small molecule PBD inhibitors exhibit poor cellular efficacy and/or selectivity. Here, we report structure-activity relationship (SAR) studies on triazoloquinazolinone-derived inhibitors, such as 43 (a 1-thioxo-2,4-dihydrothieno[2,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one) that effectively block Plk1, but not Plk2 and Plk3 PBDs, with improved affinity and drug-like properties. The range of prodrug moieties needed for thiol group masking of the active drugs has been expanded to increase cell permeability and mechanism-based cancer cell (L363 and HeLa) death. For example, a 5-thio-1-methyl-4-nitroimidazolyl prodrug 80, derived from 43, showed an improved cellular potency (GI50 4.1 μM). As expected, 80 effectively blocked Plk1 from localizing to centrosomes and kinetochores and consequently induced potent mitotic block and apoptotic cell death. Another prodrug 78 containing 9-fluorophenyl in place of the thiophene-containing heterocycle in 80 also induced a comparable degree of anti-Plk1 PBD effect. However, orally administered 78 was rapidly converted in the bloodstream to parent drug 15, which was shown be relatively stable toward in vivo oxidation due to its 9-fluorophenyl group in comparison to unsubstituted phenyl. Further derivatization of these inhibitors, particularly to improve the systemic prodrug stability, could lead to a new class of therapeutics against Plk1-addicted cancers.