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As a key regulator for hormone activity, human aldo-keto reductase family 1 member C3 (AKR1C3) plays crucial roles in the occurrence of various hormone-dependent or independent malignancies. It is a promising target for treating castration-resistant prostate cancer (CRPC). However, the development of AKR1C3 specific inhibitors remains challenging due to the high sequence similarity to its isoform AKR1C2. Here, we performed a combined in silico study to illuminate the inhibitory preference of 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acids for AKR1C3 over AKR1C2, of which compound 38 can achieve up to 5000-fold anti-AKR1C3 selectivity. Our umbrella sampling (US) simulations together with end-point binding free energy calculation MM/GBSA uncover that the high inhibition selectivity originates from the different binding modes, namely "Inward" and "Outward," of this compound series in AKR1C3 and AKR1C2, respectively. In AKR1C3/38, the tetrahydroquinoline moiety of 38 is accommodated inside the SP1 pocket and interacts favorably with surrounding residues, while, in AKR1C2/38, the SP1 pocket is too small to hold the bulky tetrahydroquinoline group that instead moves out of the pocket with 38 transitioning from an "Inward" to an "Outward" state. Further 3D-QSAR and energy decomposition analyses suggest that SP1 in AKR1C3 prefers to bind with a rigid bicyclic moiety and the modification of the R

[Image: see text] The bromodomain and extra-terminal (BET) domain family of proteins, which include its prototypical member Brd4, is implicated in a variety of cancers and viral infections due to their interaction with cellular and viral proteins. BET proteins contain two bromodomains, a common protein motif that selectively binds acetylated lysine on histones. However, they are structurally distinct from other bromodomain-containing proteins because they encode a unique C-terminal extra-terminal (ET) domain that is important for the protein–protein interactions including jumonji C-domain-containing protein 6 (JMJD6) and histone–lysine N-methyltransferase NSD3 (NSD3). Brd4 functions primarily during transcription as a passive scaffold linking cellular and viral proteins to chromatin. The rapid development of clinical inhibitors targeting Brd4 highlights the importance of this protein as an anticancer target. Current therapeutic approaches focus on the development of small molecule acetylated lysine mimics of histone marks that block the ability of the bromodomains to bind their chromatin marks. Thus far, bromodomain-targeted agents have shown dose-limiting toxicities due to off-target effects on other bromodomain-containing proteins. Here, we exploited a viral–host protein interaction interface to design peptides for the disruption of BET protein function. A murine leukemia virus (MLV) integrase-derived peptide (ET binding motif, EBM) and its shorter minimal binding motif (pentapeptide LKIRL) were sufficient to directly bind the Brd4 ET domain and reduce cellular proliferation of an acute myeloid leukemia cell line. Using computational and biochemical approaches, we identified the minimal essential contacts between EBM and LKIRL peptides and the Brd4 ET domain. Our findings provide a structural foundation for inhibiting BET/Brd4-mediated cancers by targeting the ET domain with small peptide-based inhibitors.

A facile chemical solution deposition method was used to prepare La1−xAgxMnO3 thin films with 0.1 ≤ x ≤ 0.3 on LaAlO3 (001) single-crystal substrates. It was observed that the prepared thin films were grown epitaxially on the substrates with the epitaxial relationships of La1−xAgxMnO3(001) ||LaAlO3 (001) and La1−xAgxMnO3[110]||LaAlO3[110]. The lattice constant was decreased firstly and then saturated with doping. Both the ferromagnetic and semiconductor-metallic transition temperatures were firstly enhanced and then tended to saturate with Ag doping. The La0.85Ag0.15MnO3 showed the highest ferromagnetic transition temperature of 281 K and semiconductor-metallic transition temperature of 300 K. The results will provide a facile method to deposit epitaxial Ag-doped LaMnO3 thin films with potential room temperature applications.

Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime–based therapies whose success rates are currently limited. The pyridinium cation hampers uptake of OPs into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are nonproductive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)–inhibited human AChE, here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-human AChE conjugates. Oxime group reorientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, “smart” accelerated antidotes against OP toxicity.

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.

An androgen receptor (AR) is an intensively studied treatment target for castration-resistant prostate cancer that is irresponsive to conventional antiandrogen therapeutics. Binding function 3 (BF3) inhibitors with alternative modes of action have emerged as a promising approach to overcoming antiandrogen resistance. However, how these BF3 inhibitors modulate AR function remains elusive, hindering the development of BF3-targeting agents. Here, we performed an integrated computational study to interrogate the binding mechanism of several known BF3 inhibitors with ARs. Our results show that the inhibitory effect of the BF3 antagonists arises from their allosteric modulation of the activation function (AF2) site, which alters the dynamic coupling between the BF3 and AF2 sites as well as the AF2-coactivator (SRC2-3) interaction. Moreover, the per-residue binding energy analyses reveal the "anchor" role of the linker connecting the phenyl ring and benzimidazole/indole in these BF3 inhibitors. Furthermore, the allosteric driver-interacting residues are found to include both "positive", e.g., Phe673 and Asn833, and "negative" ones, e.g., Phe826, and the differential interactions with these residues provide an explanation why stronger binding does not necessarily result in higher inhibitory activities. Finally, our allosteric communication pathway analyses delineate how the allosteric signals triggered by BF3 binding are propagated to the AF2 pocket through multiple short- and/or long-ranged transmission pathways. Collectively, our combined computational study provides a comprehensive structural mechanism underlying how the selected set of BF3 inhibitors modulate AR function, which will help guide future development of BF3 antagonists.

Nucleophilic oxime reactivators of organophosphate (OP) inhibited human acetylcholinesterase (hAChE) are reactivating antidotes against OP intoxication from nerve agent (e.g., VX, sarin) or OP pesticide (e.g., paraoxon) exposure. Within the past decade it has become increasingly clear that for effective and complete recovery from OP intoxication antidotal action is needed in both peripheral and central nervous system tissues. We are using structure-based design to create uncharged bis-oxime reactivators that possess superior in vitro properties for reactivation of various OP-conjugates of hAChE compared to the known charged pyridinium aldoxime-based therapies. Our strategies involve studying hAChE from the atomic to the molecular level by employing a combination of experimental and theoretical techniques, including X-ray and neutron crystallography, neutron vibrational spectroscopy and molecular simulations. Starting with RS-170B, MMB4 (both charged) and RS194B (uncharged) oximes we have obtained a clear view of their binding to native and VX- or paraoxon-inhibited hAChE. Neutron vibrational spectra in the 5-50 cm-1 frequency regime indicated softening of the vibrational dynamics in the paraoxon-bound hAChE, pointing to significant softening of picosecond vibrational motions of the acyl pocket loop that must accommodate one of the pesticide’s ethoxy groups. We have used our crystal structures to design and test uncharged bis-oximes, demonstrating in vitro that several of them exceeded efficacy of RS194B in reactivation of sarin, cyclosarin, VX and paraoxon inhibited hAChE. To further the reactivator design, we are working on obtaining neutron structures of hAChE which will provide direct experimental observation of hydrogen atoms to determine protonation states of hAChE residues and bound reactivators. Our designed uncharged bis-oxime antidotes are a conceptually novel, effective scaffold of nucleophilic reactivators and a promising new resource for creation of adjustable, accelerated centrally active antidotes against OP intoxication.

A series of novel pyraclostrobin derivatives were designed and prepared as antifungal agents. Their antifungal activities were tested in vitro with five important phytopathogenic fungi, namely, Batrylis cinerea, Phytophthora capsici, Fusarium sulphureum, Gloeosporium pestis and Sclerotinia sclerotiorum using the mycelium growth inhibition method. Among these compounds, 5s displayed IC50 value of 0.57 μg/mL against Batrylis cinerea and 5k-II displayed IC50 value of 0.43 μg/mL against Sclerotinia sclerotiorum, which were close to that of the positive control pyraclostrobin (0.18 μg/mL and 0.15 μg/mL). Other compounds 5f, 5k-II, 5j, 5m and 5s also exhibited strong antifungal activity. Further enzymatic assay demonstrated compound 5s inhibited porcine bc1 complex with IC50 value of 0.95 μM. The statistical results from an integrated computational pipeline demonstrated the predicted total binding free energy for compound 5s is the highest. Consequently, compound 5s with the biphenyl-4-methoxyl side chain could serve as a new motif as inhibitors of bc1 complex and deserve to be further investigated.

Exposure to organophosphorus compounds (OPs) may be fatal if untreated, and a clear and present danger posed by nerve agent OPs has become palpable in recent years. OPs inactivate acetylcholinesterase (AChE) by covalently modifying its catalytic serine. Inhibited AChE cannot hydrolyze the neurotransmitter acetylcholine leading to its build-up at the cholinergic synapses and creating an acute cholinergic crisis. Current antidotes, including oxime reactivators that attack the OP-AChE conjugate to free the active enzyme, are inefficient. Better reactivators are sought, but their design is hampered by a conformationally rigid portrait of AChE extracted exclusively from 100K X-ray crystallography and scarcity of structural knowledge on human AChE (hAChE). Here, we present room temperature X-ray structures of native and VX-phosphonylated hAChE with an imidazole-based oxime reactivator, RS-170B. We discovered that inhibition with VX triggers substantial conformational changes in bound RS-170B from a “nonproductive” pose (the reactive aldoxime group points away from the VX-bound serine) in the reactivator-only complex to a “semi-productive” orientation in the VX-modified complex. This observation, supported by concurrent molecular simulations, suggested that the narrow active-site gorge of hAChE may be significantly more dynamic than previously thought, allowing RS-170B to reorient inside the gorge. Furthermore, we found that small molecules can bind in the choline-binding site hindering approach to the phosphorous of VX-bound serine. Our results provide structural and mechanistic perspectives on the reactivation of OP-inhibited hAChE and demonstrate that structural studies at physiologically relevant temperatures can deliver previously overlooked insights applicable for designing next-generation antidotes.

Prostate cancer is the most common cancer in men and it is estimated that 32,000 men in the US alone will die of this cancer in 2019. Androgen deprivation therapy (ADT) has been the mainstay of prostate cancer therapy. However, most of the patients will progress to castration resistance prostate cancer (CRPC) after several years of treatment. The current treatment of CRPC includes chemotherapeutic agents (docetaxel) and agents that target androgen signaling (abiraterone, enzalutamide and apalutamide). Ultimately, however, most patients develop resistance to the treatments. Two of the mechanisms of resistance involve the expression of constitutively active androgen receptor variant (AR-V7) and the increase in intra-tumoral biosynthesis of androgens. Several treatment strategies targeting AR-V7 have been reported. For the intra-tumoral biosynthesis of androgens, one of the enzymes, AKR1C3, plays a critical role in the development of CRPC and has been reported to be consistently elevated in CRPC patients who are resistant to abiraterone and enzalutamide. We have been involved in the development of agents to treat CRPC and overcome drug resistance of CRPC patients. We reported that niclosamide, an FDA-approved anthelminthic agent, could degrade AR-V7. We also reported that niclosamide, as well as an AKR1C3 inhibitor, indomethacin, were able to overcome enzalutamide and abiraterone resistance in several prostate cancer cell lines, suggesting that niclosamide is also an AKR1C3 inhibitor. By examining the reported structures of AKR1C3 inhibitors, niclosamide was found to contain the core scaffold of 4-Hydroxy-1-(4-methoxybenzyl)-N-(3-(trifluoromethyl) phenyl)-1H-1,2,3-triazole-5-carboxamide. Using this knowledge, we designed a series compounds with dual AR degrading and AKR1C3 inhibitory activities. Preliminary studies showed the compounds caused AR-V7 degradation and to a lesser extent AR full length degradation. The compounds also inhibited AKR1C3 and caused a dose dependent growth inhibition of several prostate cancer cell lines. Citation Format: Enming Xing, Xiaotian Kong, Wei Lou, Ruihua Shi, Xiaolin Cheng, Allen C. Gao, Pui Kai Li. Design, synthesis and study of small molecules with both AR degradation and AKR1C3 inhibitory activities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5681.

Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake of OPs into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are nonproductive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE, here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved

Androgen receptor (AR), as a member of the nuclear receptor (NR) superfamily, regulates the gene transcription in response to the sequential binding of diverse agonists and coactivators. Great progress has been made in studies on the pharmacology and structure of AR, but the atomic level mechanism of the bidirectional communications between the ligand-binding pocket (LBP) and the activation function-2 (AF2) region of AR remains poorly understood. Therefore, in this study, molecular dynamics (MD) simulations and free energy calculations were carried out to explore the interactions among water, agonist (DHT) or antagonist (HFT), AR, and coactivator (SRC3). Upon the binding of an agonist (DHT) or antagonist (HFT), the LBP structure would transform to the agonistic or antagonistic state, and the conformational changes of the LBP would regulate the structure of the AF2 surface. As a result, the binding of the androgen DHT could promote the recruitment of the coactivator SRC3 to the AF2, and on the contrary, the binding of the antagonist HFT would induce a perturbation to the shape of the AF2 and then weaken its accommodating capability of the coactivators with the LXXLL motif. The simulation results illustrated that the DHT-AR binding affinity was enhanced by the association of the coactivator SRC3, which would reduce the conformational fluctuation of the AR-LBD and expand the size of the AR LBP. On the other hand, the coactivator-to-HFT allosteric pathway, which involves the SRC3, helix 3 (H3), helix 4 (H4), the loop (L1-3) between helix 1 (H1) and H3, and HFT, was characterized. The HFT's skewness and different interactions between HFT and the LBP were observed in the SRC3-present AR. The mutual communications between the AF2 surface and LBP, together with the processes involving the interplay of the ligand binding and coactivator recruitment events, would help in understanding the association of coactivators and rationally develop potent drugs to inhibit the activity of AR.

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.

Parabens have been widely used in packaged foods, pharmaceuticals, and personal-care products. Considering their potential hydrolysis, we herein investigated structural features leading to the disruption of human androgen receptor (AR) and whether hydrolysis could alleviate such effects using the recombinant yeast two-hybrid assay. Parabens with an aryloxy side chain such as benzyl paraben and phenyl paraben have the strongest antiandrogenic activity. The antiandrogenic activity of parabens with alkyloxyl side chains decreases as the side chain length increases from 1 to 4, and no antiandrogenic effect occurred for heptyl, octyl, and dodecyl parabens with the number of alkoxyl carbon atoms longer than 7. The antiandrogenic activity of parabens correlates significantly with their binding energies (R2 = 0.84, p = 0.01) and were completely diminished after the hydrolysis, particularly for parabens with aryloxy side chains. The Km for the hydrolysis of parabens with aromatic moiety side chain is 1 order of ma...

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.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows insensitivity towards many chemotherapeutic drugs and target-based drugs. There is an urgently need to identify PDAC disease mechanism and detect novelty drug targets. With the large availability of protein interaction networks and microarray data supported, to identify the disease essential genes that have biological significance for potential drug targets is a challenge issue still. Methods: In this study, protein interaction network PathPPI, DrugBank targets and 374 transcriptome profiles from Gene Expression Omnibus (GEO) by Affymetrix HU 133 Plus 2.0 array test are collected. These microarray data includes 140 PDAC, 92 pancreatic cancer cell-line, 58 human normal pancreas tissues, and 72 patient original xenograft mouse. Based on these datasets, a novelty-integrated algorithm was developed for drug target prioritization by perturbed gene expression and network information. The integration network included data driven reconstruction network of gene regulatory (GRN) by Bayesian Network and protein interaction PathPPI network. The perturbed gene expression was refer to the differential expression genes of tumor verse adjacent normal. A novel hybrid method based on genetic algorithm (GA) is proposed to search the attractor with the maximum network perturbation for candidate drug targets in PDAC.These targets’ molecular mechanism was revealed further by network structure comparison of PDAC that is consistent with xerograph mouse model and cancer cells. Results: Highly accordance 56 genes with high perturbation score both in mRNAs and protein-protein network were identified and recommended as candidate drug targets for PDAC, including 16 novel targets, such as PSMD2, EPOR and PSMB9. Fifty-four essential genes shown strong concordance among tumor-model, patient drive xenograft mouse and pancreatic cancer cell-line by systematically comparison. We identified 1375 dysregulated genes that enriched in 25 pathways in PDAC by Gene Set Enrichment Analysis, among which 244 genes played as hubs in reconstructed PDAC regulatory network. Conclusion: In the study, we developed a global optimization-based inference of network perturbation to detect attractor for drug-target identification in PDAC tumors. The assembling Bayesian Network-based approach with protein-protein interaction provides a comprehensive information to observe energy transfer of gene perturbation in network to detect global optimum attractor for drug target selection. Citation Format: Lijun Cheng, Enze Liu, Li Lang, Xiaolin Cheng, Xiaotian Kong, Korc Murray. Integrated network analysis reveals potentially novel molecular pathways mechanism and therapeutic targets of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4257.

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.

Due to the high sequence identity of the binding pockets of cyclin-dependent kinases (CDKs), designing highly selective inhibitors towards a specific CDK member remains a big challenge. 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine derivatives are effective inhibitors of CDKs, among which the most promising inhibitor 12u demonstrates high binding affinity to CDK9 and attenuated binding affinity to other homologous kinases, such as CDK2. In this study, in order to rationalize the principle of the binding preference towards CDK9 over CDK2 and to explore crucial information that may aid the design of selective CDK9 inhibitors, MM/GBSA calculations based on conventional molecular dynamics (MD) simulations and enhanced sampling simulations (umbrella sampling and steered MD simulations) were carried out on two representative derivatives (12u and 4). The calculation results show that the binding specificity of 12u to CDK9 is primarily controlled by conformational change of the G-loop and variation of the van der Waals interactions. Furthermore, the enhanced sampling simulations revealed the different reaction coordinates and transient interactions of inhibitors 12u and 4 as they dissociate from the binding pockets of CDK9 and CDK2. The physical principles obtained from this study may facilitate the discovery and rational design of novel and specific inhibitors of CDK9.

Angiopoietin (ANG) ligands and their downstream TIE receptors have been validated as the second vascular signaling system involving vessel remodeling and maturation. Among them, the ANG/TIE-2 signaling pathway is involved in numerous life-threatening diseases and has become an attractive potential therapeutic target. Several large-molecule inhibitors targeting the ANG/TIE-2 axis have recently entered clinical phase for the therapy of various solid tumors, but selective small-molecule inhibitors of TIE-2 are still quite limited. In the present work, structure-based virtual screening was performed to search for type-I inhibitors of TIE-2. Of the only 41 compounds selected by our strategy, 8 molecules with the concentration of 25 μg/mL exhibit over 50% inhibitory rate against TIE-2 in in vitro enzymatic activity assay, and the IC50 values of 2 hits are lower than 1 μM. Further optimization and SAR analysis based on compound TP-S1-30 and 31 were carried out by using substructure searching strategy, leading to the discovery of several sub-100 nM inhibitors. Among them, the most potent compound, TP-S1-68, showed an inhibitory IC50 of 0.149 μM. These novel inhibitors of TIE-2 discovered in this study and the analogs of the active core scaffolds can serve as the starting points for further drug development.

Anaplastic lymphoma kinase (ALK) has gained increased attention as an attractive therapeutic target for the treatment of various cancers, especially non-small-cell lung cancer (NSCLC). Recently, piperidine carboxamides were reported as Type I1/2 inhibitors of ALK, which occupy both the ATP binding site and the back ATP hydrophobic cavity in DFG-in conformation. Due to the dynamic behavior of ALK in the binding of Type I1/2 inhibitors, the accurate predictions of the binding structures and relative binding potencies of these inhibitors are quite challenging. In this study, different modeling techniques, including molecular docking, ensemble docking based on multiple receptor conformations, molecular dynamics simulations and free energy calculations, were utilized to explore the binding mechanisms of piperidine carboxamides. Our predictions show that the conventional docking protocols are not sufficient to predict the relative binding potencies of the studied inhibitors with high accuracy, but incorporating protein flexibility before or after docking is quite effective to improve the prediction accuracy. Notably, the binding free energies predicted by MM/GBSA or MM/PBSA based on the MD simulations for the docked poses give the highest correlation with the experimental data, highlighting the importance of the inclusion of receptor flexibility for the accurate predictions of the binding potencies for Type I1/2 inhibitors of ALK. Furthermore, the comprehensive analysis of several pairs of representative inhibitors demonstrates the importance of hydrophobic interactions in improving the binding affinities of the inhibitors with the hot-spot residues surrounding the binding pocket. This work is expected to provide valuable clues for further rational design of novel and potent Type I1/2 ALK inhibitors.