A small molecule inhibitor of PCSK9 that antagonizes LDL receptor binding via interaction with a cryptic PCSK9 binding groove.

Proprotein convertase (PC) subtilisin kexin type 9 (PCSK9) inhibits the clearance of low density lipoprotein (LDL) cholesterol from plasma by directly interacting with the LDL receptor (LDLR). As the interaction promotes elevated plasma LDL cholesterol levels and a predisposition to cardiovascular disease (CVD), it has attracted much interest as a therapeutic target. While anti-PCSK9 monoclonal antibodies have been successful in the treatment of hypercholesteremia by decreasing CVD risk, their high cost and a requirement for injection have prohibited widespread use. The advent of an orally bioavailable small molecule inhibitor of the PCSK9-LDLR interaction is an attractive alternative, however efforts have been tempered as the binding interface is unfavourable for binding by small organic molecules. Despite its challenging nature, we report herein the discovery of compound 3f as a small molecule inhibitor of PCSK9. The kinase inhibitor nilotinib emerged from a computational screen that was applied to identify compounds that may bind to a cryptic groove within PCSK9 and proximal to the LDLR-binding interface. A subsequent in vitro PCSK9-LDLR binding assay established that nilotinib was a bona fide but modest inhibitor of the interaction (IC50 = 9.8 µM). Through multiple rounds of medicinal chemistry, 3f emerged as a lead-like molecule by demonstrating disruption of the PCSK9-LDLR interaction at nanomolar levels in vitro (IC50 = 537 nM) with no inhibitory activity (IC50 > 10 µM) against a small panel of kinases. Compound 3f restored LDL uptake by liver cells at sub-micromolar levels and demonstrated excellent bioavailability when delivered subcutaneously in mice. Most significantly, compound 3f lowered total cholesterol levels in the plasma of wild-type mice, thereby providing proof-of-concept that the notion of a small molecule inhibitor against PCSK9 is therapeutically viable.

Computational Identification of Antibody Epitopes on the Dengue Virus NS1 Protein.

We have previously described a method to predict antigenic epitopes on proteins recognized by specific antibodies. Here we have applied this method to identify epitopes on the NS1 proteins of the four Dengue virus serotypes (DENV1-4) that are bound by a small panel of monoclonal antibodies 1H7.4, 1G5.3 and Gus2. Several epitope regions were predicted for these antibodies and these were found to reflect the experimentally observed reactivities. The known binding epitopes on DENV2 for the antibodies 1H7.4 and 1G5.3 were identified, revealing the reasons for the serotype specificity of 1H7.4 and 1G5.3, and the non-selectivity of Gus2. As DENV NS1 is critical for virus replication and a key vaccine candidate, epitope prediction will be valuable in designing appropriate vaccine control strategies. The ability to predict potential epitopes by computational methods significantly reduces the amount of experimental work required to screen peptide libraries for epitope mapping.

Tropomyosin1 isoforms underlie epithelial to mesenchymal plasticity, metastatic dissemination, and resistance to chemotherapy in high-grade serous ovarian cancer.

Phenotypic plasticity, defined as the ability of individual cells with stable genotypes to exert different phenotypes upon exposure to specific environmental cues, represent the quintessential hallmark of the cancer cell en route from the primary lesion to distant organ sites where metastatic colonization will occur. Phenotypic plasticity is driven by a broad spectrum of epigenetic mechanisms that allow for the reversibility of epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions (EMT/MET). By taking advantage of the co-existence of epithelial and quasi-mesenchymal cells within immortalized cancer cell lines, we have analyzed the role of EMT-related gene isoforms in the regulation of epithelial mesenchymal plasticity (EMP) in high grade serous ovarian cancer. When compared with colon cancer, a distinct spectrum of downstream targets characterizes quasi-mesenchymal ovarian cancer cells, likely to reflect the different modalities of metastasis formation between these two types of malignancy, i.e. hematogenous in colon and transcoelomic in ovarian cancer. Moreover, upstream RNA-binding proteins differentially expressed between epithelial and quasi-mesenchymal subpopulations of ovarian cancer cells were identified that underlie differential regulation of EMT-related isoforms. In particular, the up- and down-regulation of RBM24 and ESRP1, respectively, represent a main regulator of EMT in ovarian cancer cells. To validate the functional and clinical relevance of our approach, we selected and functionally analyzed the Tropomyosin 1 gene (TPM1), encoding for a protein that specifies the functional characteristics of individual actin filaments in contractile cells, among the ovarian-specific downstream AS targets. The low-molecular weight Tpm1.8/9 isoforms are specifically expressed in patient-derived ascites and promote invasion through activation of EMT and Wnt signaling, together with a broad spectrum of inflammation-related pathways. Moreover, Tpm1.8/9 expression confers resistance to taxane- and platinum-based chemotherapy. Small molecule inhibitors that target the Tpm1 isoforms support targeting Tpm1.8/9 as therapeutic targets for the development of future tailor-made clinical interventions.

Computational identification of epitopes in the glycoproteins of novel bunyavirus (SFTS virus) recognized by a human monoclonal antibody (MAb 4-5).

In this work, we have developed a new approach to predict the epitopes of antigens that are recognized by a specific antibody. Our method is based on the "multiple copy simultaneous search" (MCSS) approach which identifies optimal locations of small chemical functional groups on the surfaces of the antibody, and identifying sequence patterns of peptides that can bind to the surface of the antibody. The identified sequence patterns are then used to search the amino-acid sequence of the antigen protein. The approach was validated by reproducing the binding epitope of HIV gp120 envelop glycoprotein for the human neutralizing antibody as revealed in the available crystal structure. Our method was then applied to predict the epitopes of two glycoproteins of a newly discovered bunyavirus recognized by an antibody named MAb 4-5. These predicted epitopes can be verified by experimental methods. We also discuss the involvement of different amino acids in the antigen-antibody recognition based on the distributions of MCSS minima of different functional groups.

Discovery of 2-(alpha-methylbenzylamino) pyrazines as potent Type II inhibitors of FMS.

A series of 2-(alpha-methylbenzylamino) pyrazines have shown to be potent inhibitors of the FMS tyrosine receptor kinase. Details of SAR studies, modeling and synthesis of compounds within this series are reported.

Phenylaminopyrimidines as inhibitors of Janus kinases (JAKs).

A series of phenylaminopyrimidines has been identified as inhibitors of Janus kinases (JAKs). Development of this initial series led to the potent JAK2/JAK1 inhibitor CYT387 (N-(cyanomethyl)-4-[2-[[4-(4-morpholinyl)phenyl]amino]-4-pyrimidinyl]-benzamide). Details of synthesis and SAR studies of these compounds are reported.

Systematic comparison of empirical forcefields for molecular dynamic simulation of insulin.

The use of atomistic simulation methodologies based on empirical forcefields has enhanced our understanding of many physical processes governing protein structure and dynamics. However, the forcefields used in classical modeling studies are often designed for a particular class of proteins and rely on continuous improvement and validation by comparison of simulations with experimental data. We present a comprehensive comparison of five popular forcefields for simulating insulin. The effect of each forcefield on the conformational evolution and structural properties of the peptide is analyzed in detail and compared with available experimental results. In this study we observed that different forcefields favor different structural trends. However, the all-atom forcefield CHARMM27 and the united-atom forcefield GROMOS 43A1 delivered the best representation of the experimentally observed dynamic behavior of chain B of insulin.

Comparative study of insulin chain-B in isolated and monomeric environments under external stress.

We have conducted a series of theoretical simulations of insulin chain-B under different electric field conditions. This work extends our previous studies of the isolated chain-B by including chain-A and revealing the effects of chemical stress. For this complete protein, we observed increased stability under ambient conditions and under the application of thermal stress, compared to isolated chain-B. On the other hand, the presence of chain-A enhanced the effects of the applied electric field. Under the static field, the presence of chain-A lowered the strength of the field necessary to stretch the protein. Under the oscillating fields, there was relatively less stretching due to the competitive alignment process of the three helical regions with respect to the field. At high field strengths, we observed that the high frequency oscillating field caused less secondary structure disruption than a lower frequency field of the same strength.

Computational Prediction of the Epitopes of HA1 Protein of Influenza Viruses to its Neutralizing Antibodies.

In this work, we have used a new method to predict the epitopes of HA1 protein of influenza virus to several antibodies HC19, CR9114, BH151 and 4F5. While our results reproduced the binding epitopes of H3N2 or H5N1 for the neutralizing antibodies HC19, CR9114, and BH151 as revealed from the available crystal structures, additional epitopes for these antibodies were also suggested. Moreover, the predicted epitopes of H5N1 HA1 for the newly developed antibody 4F5 are located at the receptor binding domain, while previous study identified a region 76-WLLGNP-81 as the epitope. The possibility of antibody recognition of influenza virus via different mechanism by binding to different epitopes of an antigen is also discussed.

On-target action of anti-tropomyosin drugs regulates glucose metabolism.

The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 ß-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1's function with few off-target effects.

Effect of frequency on insulin response to electric field stress.

There are many unanswered questions regarding the precise way in which proteins respond to external stress. Since the function of proteins is critically linked to their three-dimensional structures, exposure to any form of stress which may induce changes in conformation can potentially initiate severe cellular dysfunction. This is particularly relevant with regard to the increasing presence of electromagnetic devices in today's environment and the possible effects on human health. Previously, we investigated the effect of electric field of various strengths on insulin chain-B under static and oscillating conditions. This paper expands on our previous work by subjecting the peptide to an oscillating electric field of different frequencies. We observed a frequency-dependent effect where the application of lower-frequency oscillating fields resulted in static-field-like behavior of the peptide, whereby the intrinsic flexibility of the protein is constrained, thus potentially restricting access to the protein's active state.

Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II.

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of "turns" in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.

Identification of Cancer-Targeted Tropomyosin Inhibitors and Their Synergy with Microtubule Drugs.

Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2-M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo This combination warrants further study. Mol Cancer Ther; 16(8); 1555-65. ©2017 AACR.

Designing amino acid residues with single-conformations.

Drug design can benefit from the use of non-coded amino acids, such as alpha-amino isobutyric acids (Aib) or sarcosine (N-methyl-glycine). Non-coded amino acids can confer resistance to enzymatic degradation and increase the conformational stability of the peptides. We have simulated the conformational effects of combining N-methylation, bulky groups on the Calpha atom and/or thioamides using the class II CFF91 force field and our thioamide force field parameters. Although single amino acid substitutions (e.g. Aib) can restrict the available conformations, they do not necessarily lead to unique conformers, however, we predict that some of the amino acids described in this report will fold to a single phi, psi conformation (e.g. N-methylated and thioamide penicillamine). Several other amino acid/thiopeptide combinations were designed, which are predicted to prefer only two conformations. Novel amino acids of this type should prove useful for designing peptides with defined conformations.

Electric field effects on insulin chain-B conformation.

The response of proteins to different forms of stress continues to be a topic of major interest, especially with the proliferation of electromagnetic devices conjectured to have detrimental effects on human health. In this paper, we have performed molecular dynamics simulations on insulin chain-B under the influence of both static and oscillating electric fields, ranging from 10(7) to 10(9) V/m. We have found that both variants have an effect on the normal behavior of the protein, with oscillating fields being more disruptive to the structure as compared to static fields of similar effective strength. The application of a static field had a stabilizing effect on the secondary structure, restricting the inherent flexibility that is crucial for insulin's biological activity.

Modeling the epidermal growth factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand binding process.

Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of ligands such as EGF or transforming growth factor alpha (TGF-alpha) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10-16, 36-37, 40-47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-alpha which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.

Antibody recognition of Shiga toxins (Stxs): computational identification of the epitopes of Stx2 subunit A to the antibodies 11E10 and S2C4.

We have recently developed a new method to predict the epitopes of the antigens that are recognized by a specific antibody. In this work, we applied the method to identify the epitopes of the Shiga toxin (Stx2 subunit A) that were bound by two specific antibodies 11E10 and S2C4. The predicted epitopes of Stx2 binding to the antibody 11E10 resembles the recognition surface constructed by the regions of Stx2 identified experimentally. For the S2C4, our results indicate that the antibody recognizes the Stx2 at two different regions on the protein surface. The first region (residues 246-254: ARSVRAVNE) is similar to the recognition region of the 11E10, while the second region is formed by two epitopes. The second region is particularly significant because it includes the amino acid sequence region that is diverse between Stx2 and other Stx (residues 176-188: QREFRQALSETAPV). This new recognition region is believed to play an important role in the experimentally observed selectivity of S2C4 to the Stx2.

A novel class of anticancer compounds targets the actin cytoskeleton in tumor cells.

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

The structural basis of Janus kinase 2 inhibition by a potent and specific pan-Janus kinase inhibitor.

JAK2, a member of the Janus kinase (JAK) family of protein tyrosine kinases (PTKs), is an important intracellular mediator of cytokine signaling. Mutations of the JAK2 gene are associated with hematologic cancers, and aberrant JAK activity is also associated with a number of immune diseases, including rheumatoid arthritis. Accordingly, the development of JAK2-specific inhibitors has tremendous clinical relevance. Critical to the function of JAK2 is its PTK domain. We report the 2.0 A crystal structure of the active conformation of the JAK2 PTK domain in complex with a high-affinity, pan-JAK inhibitor that appears to bind via an induced fit mechanism. This inhibitor, the tetracyclic pyridone 2-tert-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-1, was buried deep within a constricted ATP-binding site, in which extensive interactions, including residues that are unique to JAK2 and the JAK family, are made with the inhibitor. We present a structural basis of high-affinity JAK-specific inhibition that will undoubtedly provide an invaluable tool for the further design of novel, potent, and specific therapeutics against the JAK family.

Effects of thioamide substitutions on the conformation and stability of alpha- and 3(10)-helices.

Thiopeptides, formed by replacing the amide oxygen atom with a sp(2) sulfur atom, are useful in protein engineering and drug design because they confer resistance to enzymatic degradation and are predicted to be more rigid. This report describes our free molecular dynamics simulations with explicit water and free energy calculations on the effects of thio substitutions on the conformation of alpha-helices, 3(10)-helices, and their relative stability. The most prominent structural effect of thio substitution is the increase in the hydrogen bond distance from 2.1 A for normal peptides to 2.7 A for thiopeptides. To accommodate for the longer C[double bond]S...H-N hydrogen bond, the (phi, psi) dihedral angles of the alpha-helix changed from (-66 degrees, -42 degrees) to (-68 degrees, -38 degrees), and the rise per turn increased from 5.5 to 6.3 A. For 3(10)-helices, the (phi, psi) dihedral angles (-60 degrees, -20 degrees) and rise per turn (6.0 A) changed to (-66 degrees, -12 degrees) and 6.8 A, respectively. In terms of relative stability, the most prominent change upon thio substitution is the decrease in the free energy difference, Delta A(alpha --> 3(10)), from 14 to 3.5 kcal/mol. Therefore, normal peptides are less likely to form 3(10)-helix than are thiopeptides. Component analysis of the Delta A(alpha --> 3(10)) reviews that the entropy advantage of the 3(10)-helix for both Ac-Ala(10)-NHMe and Act-Alat(10)-NHMe is attributed to the 3(10)-helix being more flexible than the alpha-helix. Interestingly, upon thio substitution, this differential flexibility is even more apparent because the alpha-helix conformation of Act-Alat(10)-NHMe becomes more rigid due to the bulkier sulfur atom.