Discovery, X-ray Crystallography and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease.

Flaviviruses, including dengue, West Nile and recently emerged Zika virus, are important human pathogens, but there are no drugs to prevent or treat these viral infections. The highly conserved Flavivirus NS2B-NS3 protease is essential for viral replication and therefore a drug target. Compound screening followed by medicinal chemistry yielded a series of drug-like, broadly active inhibitors of Flavivirus proteases with IC50 as low as 120 nM. The inhibitor exhibited significant antiviral activities in cells (EC68: 300-600 nM) and in a mouse model of Zika virus infection. X-ray studies reveal that the inhibitors bind to an allosteric, mostly hydrophobic pocket of dengue NS3 and hold the protease in an open, catalytically inactive conformation. The inhibitors and their binding structures would be useful for rational drug development targeting Zika, dengue and other Flaviviruses.

Discovery, Structure-Activity Relationship and In Vitro Anticancer Activity of Small-Molecule Inhibitors of the Protein-Protein Interactions between AF9/ENL and AF4 or DOT1L.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause 5-10% acute leukemias with poor clinical outcomes. Protein-protein interactions (PPI) between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are drug targets for MLL-rearranged (MLL-r) leukemia. Several benzothiophene-carboxamide compounds were identified as novel inhibitors of these PPIs with IC50 values as low as 1.6 µM. Structure-activity relationship studies of 77 benzothiophene and related indole and benzofuran compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl substituent is essential for the activity. The inhibitors suppressed expression of MLL target genes HoxA9, Meis1 and Myc, and selectively inhibited proliferation of MLL-r and other acute myeloid leukemia cells with EC50 values as low as 4.7 µM. These inhibitors are useful chemical probes for biological studies of AF9/ENL, as well as pharmacological leads for further drug development against MLL-r and other leukemias.

Scaffold tailoring by a newly detected Pictet-Spenglerase activity of strictosidine synthase: from the common tryptoline skeleton to the rare piperazino-indole framework.

The Pictet-Spenglerase strictosidine synthase (STR1) has been recognized as a key enzyme in the biosynthesis of some 2000 indole alkaloids in plants, some with high therapeutic value. In this study, a novel function of STR1 has been detected which allows for the first time a simple enzymatic synthesis of the strictosidine analogue 3 harboring the piperazino[1,2-a]indole (PI) scaffold and to switch from the common tryptoline (hydrogenated carboline) to the rare PI skeleton. Insight into the reaction is provided by X-ray crystal analysis and modeling of STR1 ligand complexes. STR1 presently provides exclusively access to 3 and can act as a source to generate by chemoenzymatic approaches libraries of this novel class of alkaloids which may have new biological activities. Synthetic or natural monoterpenoid alkaloids with the PI core have not been reported before.

A proteolysis-targeting chimera molecule selectively degrades ENL and inhibits malignant gene expression and tumor growth.

BACKGROUND: Chromosome translocations involving mixed lineage leukemia 1 (MLL1) cause acute leukemia in most infants and 5-10% children/adults with dismal clinical outcomes. Most frequent MLL1-fusion partners AF4/AFF4, AF9/ENL and ELL, together with CDK9/cyclin-T1, constitute super elongation complexes (SEC), which promote aberrant gene transcription, oncogenesis and maintenance of MLL1-rearranged (MLL1-r) leukemia. Notably, ENL, but not its paralog AF9, is essential for MLL1-r leukemia (and several other cancers) and therefore a drug target. Moreover, recurrent ENL mutations are found in Wilms tumor, the most common pediatric kidney cancer, and play critical roles in oncogenesis. METHODS: Proteolysis-Targeting Chimera (PROTAC) molecules were designed and synthesized to degrade ENL. Biological activities of these compounds were characterized in cell and mouse models of MLL1-r leukemia and other cancers. RESULTS: Compound 1 efficiently degraded ENL with DC50 of 37 nM and almost depleted it at ~ 500 nM in blood and solid tumor cells. AF9 (as well as other proteins in SEC) was not significantly decreased. Compound 1-mediated ENL reduction significantly suppressed malignant gene signatures, selectively inhibited cell proliferation of MLL1-r leukemia and Myc-driven cancer cells with EC50s as low as 320 nM, and induced cell differentiation and apoptosis. It exhibited significant antitumor activity in a mouse model of MLL1-r leukemia. Compound 1 can also degrade a mutant ENL in Wilms tumor and suppress its mediated gene transcription. CONCLUSION: Compound 1 is a novel chemical probe for cellular and in vivo studies of ENL (including its oncogenic mutants) and a lead compound for further anticancer drug development.

Structure-activity relationship and antitumor activity of 1,4-pyrazine-containing inhibitors of histone acetyltransferases P300/CBP.

Acetylation of histone lysine residues by histone acetyltransferase (HAT) p300 and its paralog CBP play important roles in gene regulation in health and diseases. The HAT domain of p300/CBP has been found to be a potential drug target for cancer. Compound screening followed by structure-activity relationship studies yielded a novel series of 1,4-pyrazine-containing inhibitors of p300/CBP HAT with their IC50s as low as 1.4 µM. Enzyme kinetics and other studies support the most potent compound 29 is a competitive inhibitor of p300 HAT against the substrate histone. It exhibited a high selectivity for p300 and CBP, with negligible activity on other classes of HATs in human. Compound 29 inhibited cellular acetylation of several histone lysine residues and showed strong activity against proliferation of a panel of solid and blood cancer cells. These results indicate it is a novel pharmacological lead for drug development targeting these cancers as well as a useful chemical probe for biological studies of p300/CBP.

The Pictet-Spengler reaction in nature and in organic chemistry.

Alkaloids are an important class of natural products that are widely distributed in nature and produced by a large variety of organisms. They have a wide spectrum of biological activity and for many years were used in folk medicine. These days, alkaloids also have numerous applications in medicine as therapeutic agents. The importance of these natural products in inspiring drug discovery programs is proven and, therefore, their continued synthesis is of significant interest. The condensation discovered by Pictet and Spengler is the most important method for the synthesis of alkaloid scaffolds. The power of this synthesis method has been convincingly proven in the construction of stereochemicaly and structurally complex alkaloids.

RLStereo: Real-Time Stereo Matching Based on Reinforcement Learning.

Many state-of-the-art stereo matching algorithms based on deep learning have been proposed in recent years, which usually construct a cost volume and adopt cost filtering by a series of 3D convolutions. In essence, the possibility of all the disparities is exhaustively represented in the cost volume, and the estimated disparity holds the maximal possibility. The cost filtering could learn contextual information and reduce mismatches in ill-posed regions. However, this kind of methods has two main disadvantages: 1) cost filtering is very time-consuming, and it is thus difficult to simultaneously satisfy the requirements for both speed and accuracy; 2) thickness of the cost volume determines the disparity range which can be estimated, and the pre-defined disparity range may not meet the demand of practical application. This paper proposes a novel real-time stereo matching method called RLStereo, which is based on reinforcement learning and abandons the cost volume or the routine of exhaustive search. The trained RLStereo makes only a few actions iteratively to search the value of the disparity for each pair of stereo images. Experimental results show the effectiveness of the proposed method, which achieves comparable performances to state-of-the-art algorithms with real-time speed on the public large-scale testset, i.e., Scene Flow.

Small-molecule inhibitor of AF9/ENL-DOT1L/AF4/AFF4 interactions suppresses malignant gene expression and tumor growth.

Chromosome translocations involving mixed lineage leukemia (MLL) gene cause acute leukemia with a poor prognosis. MLL is frequently fused with transcription cofactors AF4 (~35%), AF9 (25%) or its paralog ENL (10%). The AHD domain of AF9/ENL binds to AF4, its paralog AFF4, or histone-H3 lysine-79 (H3K79) methyltransferase DOT1L. Formation of AF9/ENL/AF4/AFF4-containing super elongation complexes (SEC) and the catalytic activity of DOT1L are essential for MLL-rearranged leukemia. Protein-protein interactions (PPI) between AF9/ENL and DOT1L/AF4/AFF4 are therefore a potential drug target. Methods: Compound screening followed by medicinal chemistry was used to find inhibitors of such PPIs, which were examined for their biological activities against MLL-rearranged leukemia and other cancer cells. Results: Compound-1 was identified to be a novel small-molecule inhibitor of the AF9/ENL-DOT1L/AF4/AFF4 interaction with IC50s of 0.9-3.5 µM. Pharmacological inhibition of the PPIs significantly reduced SEC and DOT1L-mediated H3K79 methylation in the leukemia cells. Gene profiling shows compound-1 significantly suppressed the gene signatures related to onco-MLL, DOT1L, HoxA9 and Myc. It selectively inhibited proliferation of onco-MLL- or Myc-driven cancer cells and induced cell differentiation and apoptosis. Compound-1 exhibited strong antitumor activity in a mouse model of MLL-rearranged leukemia. Conclusions: The AF9/ENL-DOT1L/AF4/AFF4 interactions are validated to be an anticancer target and compound-1 is a useful in vivo probe for biological studies as well as a pharmacological lead for further drug development.

Synthesis, structure-activity relationship and antiviral activity of indole-containing inhibitors of Flavivirus NS2B-NS3 protease.

Zika virus belongs to the Flavivirus family of RNA viruses, which include other important human pathogens such as dengue and West Nile virus. There are no approved antiviral drugs for these viruses. The highly conserved NS2B-NS3 protease of Flavivirus is essential for the replication of these viruses and it is therefore a drug target. Compound screen followed by medicinal chemistry optimization yielded a novel series of 2,6-disubstituted indole compounds that are potent inhibitors of Zika virus protease (ZVpro) with IC50 values as low as 320 nM. The structure-activity relationships of these and related compounds are discussed. Enzyme kinetics studies show the inhibitor 66 most likely exhibited a non-competitive mode of inhibition. In addition, this series of ZVpro inhibitors also inhibit the NS2B-NS3 protease of dengue and West Nile virus with reduced potencies. The most potent compounds 66 and 67 strongly inhibited Zika virus replication in cells with EC68 values of 1-3 µM. These compounds are novel pharmacological leads for further drug development targeting Zika virus.

Synthesis, Structure-Activity Relationships, and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease.

Flaviviruses, including Zika, dengue, and West Nile viruses, are important human pathogens. The highly conserved NS2B-NS3 protease of Flavivirus is essential for viral replication and therefore a promising drug target. Through compound screening, followed by medicinal chemistry studies, a novel series of 2,5,6-trisubstituted pyrazine compounds are found to be potent, allosteric inhibitors of Zika virus protease (ZVpro) with IC50 values as low as 130 nM. Their structure-activity relationships are discussed. The ZVpro inhibitors also inhibit homologous proteases of dengue and West Nile viruses, and their inhibitory activities are correlated. The most potent compounds 47 and 103 potently inhibited Zika virus replication in cells with EC68 values of 300-600 nM and in a mouse model of Zika infection. These compounds represent novel pharmacological leads for drug development against Flavivirus infections.

Novel VAC14 variants identified in two Chinese siblings with childhood-onset striatonigral degeneration.

BACKGROUND: VAC14 is a component of a trimolecular complex that tightly regulates the level of phosphatidylinositol 3,5-bisphosphate [PI (3,5) P2]. VAC14 pathogenic variants cause prominent vacuolation of neurons in basal ganglia of patients with childhood-onset striatonigral degeneration (SNDC). METHODS: We identified two siblings with SNDC. Whole-exome sequencing was performed for genetic molecular analysis in these probands. RESULTS: The patients were compound heterozygotes for two novel variants in the VAC14 gene, p.Ala582Thr and p.Arg681His. The pathogenicity of these variants was indicated by a bioinformatic study and protein three-dimensional modeling. Eight previously reported SNDC cases and a Yunis-Varón syndrome caused by VAC14 mutations were summarized and compared. CONCLUSION: We present novel compound heterozygous variants (c.1744G>A/c.2042G>A) in our proband, and these novel variants were predicted to be likely pathogenic. The affected siblings were clinically severe and lethal; their phenotypes were similar to the majority of previously reported SNDC cases, with the exception of two cases that showed mild clinical manifestations. VAC14 pathogenic variants may be associated with various phenotypes. Herein, we report the Chinese siblings with SNDC, they are the first Asian cases. Our results expanded the spectrum of VAC14 pathogenic variants and the ethnic backgrounds of the affected cases.

Acetylation of histone H3K27 signals the transcriptional elongation for estrogen receptor alpha.

As approximately 70% of human breast tumors are estrogen receptor α (ERα)-positive, estrogen and ERα play essential roles in breast cancer development. By interrupting the ERα signaling pathway, endocrine therapy has been proven to be an effective therapeutic strategy. In this study, we identified a mechanism by which Transcription Start Site (TSS)-associated histone H3K27 acetylation signals the Super Elongation Complex (SEC) to regulate transcriptional elongation of the ESR1 (ERα) gene. SEC interacts with H3K27ac on ESR1 TSS through its scaffold protein AFF4. Depletion of AFF4 by siRNA or CRISPR/Cas9 dramatically reduces expression of ESR1 and its target genes, consequently inhibiting breast cancer cell growth. More importantly, a AFF4 mutant which lacks H3K27ac interaction failed to rescue ESR1 gene expression, suggesting H3K27 acetylation at TSS region is a key mark bridging the transition from transcriptional initiation to elongation, and perturbing SEC function can be an alternative strategy for targeting ERα signaling pathway at chromatin level.

Discovery, Structure-Activity Relationship, and Biological Activity of Histone-Competitive Inhibitors of Histone Acetyltransferases P300/CBP.

Histone acetyltransferase (HAT) p300 and its paralog CBP acetylate histone lysine side chains and play critical roles in regulating gene transcription. The HAT domain of p300/CBP is a potential drug target for cancer. Through compound screening and medicinal chemistry, novel inhibitors of p300/CBP HAT with their IC50 values as low as 620 nM were discovered. The most potent inhibitor is competitive against histone substrates and exhibits a high selectivity for p300/CBP. It inhibited cellular acetylation and had strong activity with EC50 of 1-3 µM against proliferation of several tumor cell lines. Gene expression profiling in estrogen receptor (ER)-positive breast cancer MCF-7 cells showed that inhibitor treatment recapitulated siRNA-mediated p300 knockdown, inhibited ER-mediated gene transcription, and suppressed expression of numerous cancer-related gene signatures. These results demonstrate that the inhibitor is not only a useful probe for biological studies of p300/CBP HAT but also a pharmacological lead for further drug development targeting cancer.

Thermodynamic investigation of DNA-binding affinity of wild-type and mutant transcription factor RUNX1.

Transcription factor RUNX1 and its binding partner CBFß play a critical role in gene regulation for hematopoiesis. Mutations of RUNX1 cause ~10% of acute myeloid leukemia (AML) with a particularly poor prognosis. The current paradigm for the leukemogenesis is that insufficient activity of wild-type (WT) RUNX1 impairs hematopoietic differentiation. The majority of mutant RUNX1 proteins lose the DNA-binding affinity and inhibit WT RUNX1 by depletion of CBFß. Here, isothermal titration calorimetry (ITC) was used to quantitatively study the interactions of WT and three clinical mutant RUNX1, CBFß and DNA. Our data show that the binding of RUNX1 to DNA is enthalpy-driven, and the affinity decreases in the order of WT > S114L > R139Q >> K83E, which support previous observations and conclusion. To find potentially beneficial RUNX1 mutations that could increase the overall RUNX1 activity, K83R and H179K mutations of RUNX1 were designed, using structure-based computational modeling, and found to possess significantly higher DNA-binding affinities than does WT RUNX1. K83R and H179K mutant RUNX1 could therefore be protein-based RUNX1 activators.

Early diagnosis improving the outcome of an infant with epileptic encephalopathy with cytoplasmic FMRP interacting protein 2 mutation: Case report and literature review.

RATIONALE: Early infantile epileptic encephalopathy (EIEE) 65 was recently shown to be caused by the cytoplasmic FMRP interacting protein 2 (CYFIP2) mutation. To date, only 5 cases have been reported in two articles, and all the outcomes in all cases were poor. PATIENT CONCERNS: In this study, we reported an 8-month-old girl with a 1 month-long history of seizures and developmental delay. Over 1 month later, she developed epileptic spasms in clusters with hypsarrhythmia on electroencephalography. DIAGNOSIS: The patient was diagnosed with EIEE 65 and trio-based whole-exome sequencing revealed a causative de novo CYFIP2 mutation c.260G >T (p.Arg87Leu). INTERVENTIONS: The proband was successively treated with multiple antiepileptic drugs, including levetiracetam, phenobarbital, VitB6, topiramate, methylprednisolone, prednisone, valproic acid and vigabatrin. OUTCOMES: After resistance to multiple anti-epileptic drugs over 2 months of treatment, she finally achieved seizure-free several days after vigabatrin administration and her developmental delay steadily improved. LESSONS: OUR: case confirmed that CYFIP2 was the pathogenic gene of EIEE 65. We also first demonstrated vigabatrin might be effective for control of seizures and helpful for the improved outcomes of these patients.

Early­onset epilepsy and microcephaly­capillary malformation syndrome caused by a novel STAMBP mutation in a Chinese boy.

To the best of our knowledge, the present study reported the case of the first Chinese patient with microcephaly­capillary malformation (MIC­CAP) syndrome caused by a novel compound heterozygous mutation in the STAMBP gene, which encodes STAM binding protein. The present study also provides a review of relevant previously published studies. A boy with MIC­CAP syndrome with developmental delay, intractable epilepsy and prominent dyskinesia was examined. A pathogenic mutation was identified by whole­exome sequencing, and the protein structure and function affected by this mutation were predicted using bioinformatics analysis. Finally, the clinical features of 16 other cases reported in previous studies were reviewed and compared. A novel compound heterozygous mutation of the STAMBP (c.1119­1G>T, c.968A>G) was identified in the present study and epilepsy was refractory, consistent with previously reported cases. The present study also highlighted the fact that STAMBP mutation­associated MIC­CAP often presents as intractable early­life epilepsy, which may lead to mortality.

Inhibition of Mutated Isocitrate Dehydrogenase 1 in Cancer.

BACKGROUND: R132H mutation of isocitrate dehydrogenase 1 (IDH1) is found in ~75% of low-grade gliomas and secondary glioblastomas as well as in several other types of cancer. More chemotypes of inhibitors of IDH1(R132H) are therefore needed. OBJECTIVE: The study aimed to develop a new class of IDH1(R132H) inhibitors as potent antitumor agents. METHOD: A biochemical assay was developed to find inhibitors of IDH1(R132H) mutant enzyme. Chemical synthesis and structure-activity relationship studies were used to find compounds with improved potency. Antitumor activities of selected compounds were evaluated. RESULTS: A series of aromatic sulfonamide compounds was found to be novel, potent inhibitors of IDH1(R132H) with Ki values as low as 0.6 µM. Structure-activity relationships of these compounds are discussed. Enzyme kinetics studies showed that one compound is a competitive inhibitor against the substrate α-KG and a non-competitive inhibitor against the cofactor NADPH. Several inhibitors were found to have no activity against wild-type IDH1, showing a high selectivity. Two potent inhibitors exhibited strong activity against proliferation of BT142 glioma cells with IDH1 R132H mutation, while these compounds did not significantly affect the growth of glioma cells without IDH1 mutation. CONCLUSION: This novel series of IDH1(R132H) inhibitors have potential to be further developed for the treatment of glioma with IDH1 mutation.

Glucocorticoids Inhibit Oncogenic RUNX1-ETO in Acute Myeloid Leukemia with Chromosome Translocation t(8;21).

Acute myeloid leukemia (AML) is a major blood cancer with poor prognosis. New therapies are needed to target oncogene-driven leukemia stem cells, which account for relapse and resistance. Chromosome translocation t(8;21), which produces RUNX1-ETO (R-E) fusion oncoprotein, is found in ~13% AML. R-E dominance negatively inhibits global gene expression regulated by RUNX1, a master transcription factor for hematopoiesis, causing increased self-renewal and blocked cell differentiation of hematopoietic progenitor cells, and eventually leukemia initiation. Methods: Connectivity-Map followed by biological activity testing were used to identify candidate compounds that can inhibit R-E-mediated gene transcription. Molecular mechanistic studies were also performed. Results: Glucocorticoid drugs, such as betamethasone and dexamethasone, were found to exhibit potent and selective in vitro and in vivo activities against R-E leukemia, as well as strong synergy when combined with chemotherapeutics. Microarray analysis showed that treatment with glucocorticoids significantly inhibited R-E's activity and reactivated that of RUNX1. Such gene expression changes caused differentiation and apoptosis of R-E leukemia cells. Our studies also show a possible molecular mechanism for the targeted therapy. Upon treatment with a glucocorticoid drug, more glucocorticoid receptor (GR) was translocated into the nucleus and bound to DNA, including promoters of RUNX1 target genes. GR was found to associate with RUNX1, but not R-E. This interaction increased binding of RUNX1 to DNA and reduced that of R-E, shifting to a RUNX1 dominance. Conclusion: Glucocorticoid drugs represent a targeted therapy for AML with chromosome translocation t(8:21). Given their high activity, favorable human pharmacokinetics as well as synergy with chemotherapeutics, glucocorticoids could be clinically useful to treat R-E AML.

Structure activity relationship and modeling studies of inhibitors of lysine specific demethylase 1.

Post-translational modifications of histone play important roles in gene transcription. Aberrant methylation of histone lysine sidechains have been often found in cancer. Lysine specific demethylase 1 (LSD1), which can demethylate histone H3 lysine 4 (H3K4) and other proteins, has recently been found to be a drug target for acute myeloid leukemia. To understand structure activity/selectivity relationships of LSD1 inhibitors, several series of cyclopropylamine and related compounds were synthesized and tested for their activities against LSD1 and related monoamine oxidase (MAO) A and B. Several cyclopropylamine containing compounds were found to be highly potent and selective inhibitors of LSD1. A novel series cyclopropylimine compounds also exhibited strong inhibitory activity against LSD1. Structure activity relationships (SAR) of these compounds are discussed. Docking studies were performed to provide possible binding models of a representative compound in LSD1 and MAO-A. Moreover, these modeling studies can rationalize the observed SARs and selectivity.

Targeting histone methylation for cancer therapy: enzymes, inhibitors, biological activity and perspectives.

Post-translational methylation of histone lysine or arginine residues plays important roles in gene regulation and other physiological processes. Aberrant histone methylation caused by a gene mutation, translocation, or overexpression can often lead to initiation of a disease such as cancer. Small molecule inhibitors of such histone modifying enzymes that correct the abnormal methylation could be used as novel therapeutics for these diseases, or as chemical probes for investigation of epigenetics. Discovery and development of histone methylation modulators are in an early stage and undergo a rapid expansion in the past few years. A number of highly potent and selective compounds have been reported, together with extensive preclinical studies of their biological activity. Several compounds have been in clinical trials for safety, pharmacokinetics, and efficacy, targeting several types of cancer. This review summarizes the biochemistry, structures, and biology of cancer-relevant histone methylation modifying enzymes, small molecule inhibitors and their preclinical and clinical antitumor activities. Perspectives for targeting histone methylation for cancer therapy are also discussed.