New Small Molecule Entry Inhibitors Targeting Hemagglutinin-Mediated Influenza A Virus Fusion

Influenza viruses are a major public health threat worldwide, and options for antiviral therapy are limited by the emergence of drug-resistant virus strains. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. Using pseudotype virus-based high-throughput screens, we have identified several new small molecules capable of inhibiting influenza virus entry. We prioritized two novel inhibitors, MBX2329 and MBX2546, with aminoalkyl phenol ether and sulfonamide scaffolds, respectively, that specifically inhibit HA-mediated viral entry. The two compounds (i) are potent (50% inhibitory concentration [IC₅₀] of 0.3 to 5.9 μM); (ii) are selective (50% cytotoxicity concentration [CC₅₀] of >100 μM), with selectivity index (SI) values of >20 to 200 for different influenza virus strains; (iii) inhibit a wide spectrum of influenza A viruses, which includes the 2009 pandemic influenza virus A/H1N1/2009, highly pathogenic avian influenza (HPAI) virus A/H5N1, and oseltamivir-resistant A/H1N1 strains; (iv) exhibit large volumes of synergy with oseltamivir (36 and 331 μM² % at 95% confidence); and (v) have chemically tractable structures. Mechanism-of-action studies suggest that both MBX2329 and MBX2546 bind to HA in a nonoverlapping manner. Additional results from HA-mediated hemolysis of chicken red blood cells (cRBCs), competition assays with monoclonal antibody (MAb) C179, and mutational analysis suggest that the compounds bind in the stem region of the HA trimer and inhibit HA-mediated fusion. Therefore, MBX2329 and MBX2546 represent new starting points for chemical optimization and have the potential to provide valuable future therapeutic options and research tools to study the HA-mediated entry process.     

Targeting c-MYC with T-Cells

Over-expression of the proto-oncogene c-MYC is frequently observed in a variety of tumors and is a hallmark of Burkitt´s lymphoma. The fact that many tumors are oncogene-addicted to c-MYC, renders c-MYC a powerful target for anti-tumor therapy. Using a xenogenic vaccination strategy by immunizing C57BL/6 mice with human c-MYC protein or non-homologous peptides, we show that the human c-MYC protein, despite its high homology between mouse and man, contains several immunogenic epitopes presented in the context of murine H2^b haplotype. We identified an MHC class II-restricted CD4⁺ T-cell epitope and therein an MHC class I-restricted CD8⁺ T-cell epitope (SSPQGSPEPL) that, after prime/boost immunization, protected up to 25% of mice against a lethal lymphoma challenge. Lymphoma-rejecting animals contained MHC multimer-binding CD8⁺ cell within the peripheral blood and displayed in vivo cytolytic activity with specificity for SSPQGSPEPL. Taken together these data suggest that oncogenic c-MYC can be targeted with specific T-cells.     

Structure-based Discovery of Novel Small Molecule Wnt Signaling Inhibitors by Targeting the Cysteine-rich Domain of Frizzled

Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC₅₀ values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled.     

Plant-Derived Small Molecule Inhibitors of Neuronal NO-Synthase: Potential Effects on Protein Degradation

Cigarette smoking is known to cause a decrease in NO production in man resulting in a variety of pathological effects, including vascular dysfunction. Aqueous extracts of cigarette and cigarette smoke contain chemical inhibitors to NO-synthases, a heme-containing cytochrome P450 enzymes. More recently, it was shown that freshly harvested leaves from the tobacco plant (Nicotiana tabacum, Solanaceae) also contain chemical inhibitors to neuronal NO-synthase (nNOS). Examination of leaves from 32 other plants representing diverse members of the plant kingdom showed that 17 other plants, besides tobacco, contain these chemical inhibitors. Of all these plants, 16 are members of the core asterids flowering plant group and 6 are members of the Solanaceae family. Although the identity of the chemicals is not known, perhaps the closely related plants contain the same or similar compounds that inhibit nNOS. The inhibitory effects are not attributable to nicotine. The discovery of these chemicals and their further characterization may help to explain the loss of nNOS in smokers. In this addendum, we discuss these results in light of the effect of tobacco-derived chemicals in inhibiting P450 cytochromes, as well as our thoughts on how the inactivation of nNOS leads to its selective downregulation through proteolytic degradation.Key Words: nitric oxide, inhibitors, tobacco, natural products, cytochrome P450, nitric oxide synthase, heat shock proteinsNO-synthase (NOS) is a cytochrome P450 like hemeprotein enzyme that catalyzes the production of NO from L-arginine.^1,2 NO is very important cellular signaling molecule, which plays a role in a variety of physiological processes including blood pressure regulation, neurotransmission, and penile erection. Like other P450 cytochromes, the catalytic site of NOS contains a heme prosthetic group that upon reduction and complexation with CO forms a chromophore that absorbs in the 450 nm region. Thus, although not closely related with respect to amino acid sequence to other P450 enzymes, the catalytic core of NOS and the chemistry involved in catalysis by NOS are highly similar to other P450 cytochromes.The cytochrome P450 enzymes comprise a very large family of enzymes that are responsible for metabolism of drugs and other xenobiotics as well as for synthesis of many hormones. There are over 2,000 P450 genes characterized in animals with over 50 genes in humans and over 1,900 genes in plants. The diversity of the P450 family is thought to reflect a complex “battle” between plants and animals.³ That is, plants are thought to have developed new alkaloids with new enzymes to protect themselves against animals whereas the animals developed new enzymes to metabolize these newly created plant toxins. This is thought to explain why the diversity in CYP genes increased dramatically approximately 400 millions years ago when organisms moved from the oceans to land.^3,4 Consistent with this hypothesis, chemicals in plants have been documented to inhibit mammalian P450 cytochromes. More importantly for humans, a variety of plant products have been documented to interact clinically with P450 cytochromes.⁵ For example, furanocoumarins found in grapefruit juice are known to enhance the oral bioavailability of felodipine, a calcium channel blocker, by inhibiting intestinal P450 metabolism of the drug.⁶In the case of tobacco-based cigarettes, hydrophobic chemicals in cigarettes have been found to inhibit P450 cytochromes. In particular, aromatase, the terminal P450 enzyme responsible for estrogen synthesis in humans, has been shown to be inhibited by tobacco smoke.⁷ Acyl derivatives of noricotine and anabasine were found to be active inhibitors of aromatase. In the case of NOS, studies by Heitzer et al.⁸ showed the amelioration of the vascular dysfunction in smokers by tetrahydrobiopterin and the authors concluded that there was a loss of endothelial NO synthesis caused by smoking. Moreover, Xie et al.⁹ showed that exposing rats to cigarette smoke led to the loss of both neuronal NO-synthase (nNOS) activity and protein. This observation reminded us of our earlier studies showing that guanabenz is a time-dependent irreversible inhibitor of nNOS that causes a loss of the penile nNOS activity and protein when given to rats.¹⁰ Thus, based on these findings, we wondered if chemicals in cigarettes could interact with NOS and explain the loss of NOS. We discovered that aqueous extracts of cigarettes and cigarette smoke could inhibit nNOS¹¹ and endothelial NOS.¹² More recently, extracts from freshly harvested tobacco leaves and leaves of various other plants from diverse phylogeny were also examined.¹³Tobacco leaves contained inhibitors to nNOS, indicating that these compounds did not necessarily arise from the curing and processing of the tobacco leaves to make cigarettes or from burning of the cigarettes. Moreover, other extracts made from other plants including 6 from the Solanacea family were found to have inhibitory activity. Thus, we believe that some common chemical or related chemicals exist that inhibits nNOS. The inhibitors are small molecules with hydrophilic and cationic qualities. In comparison, the inhibitors to aromatase are more hydrophobic than those of nNOS, likely reflecting the hydrophobic nature of the active site of aromatase, which accepts steroidal androgens. On the other hand, nNOS accepts L-arginine, a cationic water-soluble compound. The water-soluble nature of the inhibitors has so far hindered the isolation and characterization of the nNOS inhibitors, and thus we do not know the identity of these chemicals.The irreversible nature of the interaction of tobacco inhibitors with nNOS found in our studies may be important in understanding the long-term consequences of smoking. The irreversible inactivation of nNOS by various guanidine-based compounds is known to cause the enhanced proteasomal degradation of nNOS.¹⁴ The dysfunctional nNOS is known to be selectively ubiquitinated¹⁵ by a process involving Hsp70 and CHIP¹⁶ (Fig. 1). The degradation is also accelerated by inhibition of Hsp90, indicating that NOS is also regulated by the Hsp90-based chaperones.¹⁴ The loss of nNOS protein due to cigarette smoke suggests a similar process may be occurring with smokers.Open in a separate windowFigure 1Ubiquitination and Degradation of Inactivated nNOS. The nNOS is inactivated by chemicals found in some plants by alterations directed at the heme-containing active site of nNOS. This dysfunctional form of nNOS is directed for ubiquitination by an E3 ligase, such as CHIP (C-terminal Hsp-interacting protein), hsp70, and E2 ligase enzyme. The ubiquitinated nNOS is then recognized for proteasomal degradation. Closed circles represent ubiquitin molecules.The discovery of nNOS inhibitors in plants could be adequately interpreted in the context of plant-animal warfare. However, in light of the recent discovery of plant NOS,¹⁷ and in particular NOS in tobacco cells,¹⁸ it is possible that these inhibitors are endogenous modulators of the plant NOS. In this respect, an endogenous inhibitor has been identified for mammalian NOS.¹⁹ Furthermore, considering that the Hsp90-and Hsp70-based chaperones, ubiquitin, CHIP, and proteasome are found in plants and serve similar functions,^20–23 the NOS in plants may be similarly regulated. Thus, the mechanism outlined in Figure 1 for nNOS may also be pertinent to regulation of plant NOS. We believe this process reflects a fundamental biological process of protein quality control that is greatly affected by the binding of small molecules to the target protein.     

Molecular Probing of the HPV-16 E6 Protein Alpha Helix Binding Groove with Small Molecule Inhibitors

The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.     

Determination of Amino Acid Residues Responsible for Specific Interaction of Protein Kinases with Small Molecule Inhibitors

Identifying amino acid positions that determine the specific interaction of proteins with small molecule ligands, is required for search of pharmaceutical targets, drug design, and solution of other biotechnology problems. We studied applicability of an original method SPrOS (specificity projection on sequence) developed to recognize functionally significant positions in amino acid sequences. The method allows residues specific to functional subgroups to be determined within the protein family based on their local surroundings in amino acid sequences. The efficiency of the method has been estimated on the protein kinase family. The residues associated with the protein specificity to inhibitors have been predicted. The results have been verified using 3D structures of protein–ligand complexes. Three small molecule inhibitors have been tested. Residues predicted with SPrOS either in contacted the inhibitor or influenced the conformation of the ligand–binding area. Excluding close homologues from the studied set makes it possible to decrease the number of difficult to interpret positions. The expediency of this procedure was determined by the relationship between an inhibitory spectrum and phylogenic partition. Thus, the method efficiency has been confirmed by matching the prediction results with the protein 3D structures.     

Electrostatic Similarities between Protein and Small Molecule Ligands Facilitate the Design of Protein-Protein Interaction Inhibitors

One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs). We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research) SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs.     

Small Molecule Deubiquitinase Inhibitors Promote Macrophage Anti-Infective Capacity

The global spread of anti-microbial resistance requires urgent attention, and diverse alternative strategies have been suggested to address this public health concern. Host-directed immunomodulatory therapies represent one approach that could reduce selection for resistant bacterial strains. Recently, the small molecule deubiquitinase inhibitor WP1130 was reported as a potential anti-infective drug against important human food-borne pathogens, notably Listeria monocytogenes and noroviruses. Utilization of WP1130 itself is limited due to poor solubility, but given the potential of this new compound, we initiated an iterative rational design approach to synthesize new derivatives with increased solubility that retained anti-infective activity. Here, we test a small library of novel synthetic molecules based on the structure of the parent compound, WP1130, for anti-infective activity in vitro. Our studies identify a promising candidate, compound 9, which reduced intracellular growth of L. monocytogenes at concentrations that caused minimal cellular toxicity. Compound 9 itself had no bactericidal activity and only modestly slowed Listeria growth rate in liquid broth culture, suggesting that this drug acts as an anti-infective compound by modulating host-cell function. Moreover, this new compound also showed anti-infective activity against murine norovirus (MNV-1) and human norovirus, using the Norwalk virus replicon system. This small molecule inhibitor may provide a chemical platform for further development of therapeutic deubiquitinase inhibitors with broad-spectrum anti-infective activity.     

埃博拉病毒小分子抑制剂的研究进展

目前尚没有可靠的埃博拉病毒(Ebola virus,EBOV)疫苗和特异性治疗药物.2014年埃博拉病毒病在西非的爆发和肆虐警醒人类,需要加快对该病的防控研究.近几年,在EBOV小分子抑制剂的研究方面取得了较好的进展,有的已进入临床试验阶段.小分子化合物通常是针对病毒致病作用的某种机制而设计,是一个很有发展前途的研究领域.本文从抑制EBOV和其他病毒在生活周期中的穿入细胞、复制和出芽等方面综述EBOV小分子抑制剂的研究进展.     

小分子Janus 激酶抑制剂的研究与开发

近年来的研究发现,Janus 激酶(JAK) 可通过JAK-STAT 信号通路对细胞增殖、分化、凋亡以及血管生成、免疫调节等起重要作用,其抑制剂可用于骨髓纤维化、恶性肿瘤以及自身免疫性疾病的防治。简介JAK-STAT 通路与JAK2V617F 突变,分类综述用于治疗恶性血液病和肿瘤以及自身免疫性疾病与移植排斥反应的小分子JAK 抑制剂的研究与开发。     

Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the ‘DFG-out’ inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the ‘gatekeeper’ V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.     

Small Molecule Proprotein Convertase Inhibitors for Inhibition of Embryo Implantation

Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound''s lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.     

Small Molecule Screen for Candidate Antimalarials Targeting Plasmodium Kinesin-5

Plasmodium falciparum and vivax are responsible for the majority of malaria infections worldwide, resulting in over a million deaths annually. Malaria parasites now show measured resistance to all currently utilized drugs. Novel antimalarial drugs are urgently needed. The Plasmodium Kinesin-5 mechanoenzyme is a suitable “next generation” target. Discovered via small molecule screen experiments, the human Kinesin-5 has multiple allosteric sites that are “druggable.” One site in particular, unique in its sequence divergence across all homologs in the superfamily and even within the same family, exhibits exquisite drug specificity. We propose that Plasmodium Kinesin-5 shares this allosteric site and likewise can be targeted to uncover inhibitors with high specificity. To test this idea, we performed a screen for inhibitors selective for Plasmodium Kinesin-5 ATPase activity in parallel with human Kinesin-5. Our screen of nearly 2000 compounds successfully identified compounds that selectively inhibit both P. vivax and falciparum Kinesin-5 motor domains but, as anticipated, do not impact human Kinesin-5 activity. Of note is a candidate drug that did not biochemically compete with the ATP substrate for the conserved active site or disrupt the microtubule-binding site. Together, our experiments identified MMV666693 as a selective allosteric inhibitor of Plasmodium Kinesin-5; this is the first identified protein target for the Medicines of Malaria Venture validated collection of parasite proliferation inhibitors. This work demonstrates that chemical screens against human kinesins are adaptable to homologs in disease organisms and, as such, extendable to strategies to combat infectious disease.     

新型冠状病毒抗体和小分子抑制剂的研究现状

世界卫生组织已宣布新型冠状病毒感染（coronavirus disease 2019,COVID-19）的爆发为全球大流行。中和抗体和小分子抑制剂在预防及治疗COVID-19中发挥重要作用。尽管已开发出了多种中和抗体以及疫苗,但是随着病原体严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的不断变异,现有的抗体及疫苗面临巨大的挑战。小分子抑制剂主要通过干扰病毒与宿主的结合以及病毒自身的复制达到消灭病毒以及抑制病毒感染的作用,并且对SARS-CoV-2突变株具有广谱抑制作用,是当前研究的热点。近年来国内外学者对SARS-CoV-2的小分子抑制剂做了大量的研究工作,本文根据中和抗体识别的抗原表位以及小分子抑制剂的作用机制分别对用于预防及治疗COVID-19的中和抗体和小分子抑制剂进行综述,讨论其研究现状,并展望小分子抑制剂的应用前景,以期为该领域的进一步研究提供参考。     

Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.     

Analysis of Small Molecule Ligands Targeting the HIV-1 Matrix Protein-RNA Binding Site

The matrix domain (MA) of the HIV-1 precursor Gag (PrGag) protein directs PrGag proteins to assembly sites at the plasma membrane by virtue of its affinity to the phospholipid, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂). MA also binds to RNA at a site that overlaps its PI(4,5)P₂ site, suggesting that RNA binding may protect MA from associating with inappropriate cellular membranes prior to PrGag delivery to the PM. Based on this, we have developed an assay in which small molecule competitors to MA-RNA binding can be characterized, with the assumption that such compounds might interfere with essential MA functions and help elucidate additional features of MA binding. Following this approach, we have identified four compounds, including three thiadiazolanes, that compete with RNA for MA binding. We also have identified MA residues involved in thiadiazolane binding and found that they overlap the MA PI(4,5)P₂ and RNA sites. Cell culture studies demonstrated that thiadiazolanes inhibit HIV-1 replication but are associated with significant levels of toxicity. Nevertheless, these observations provide new insights into MA binding and pave the way for the development of antivirals that target the HIV-1 matrix domain.