ABSTRACT
More than fifteen million women with the human immunodeficiency virus type-1 (HIV-1) infection are of childbearing age world-wide. Due to improved and affordable access to antiretroviral therapy (ART), the number of in utero antiretroviral drug (ARV)-exposed children has exceeded a million and continues to grow. While most recommended ART taken during pregnancy suppresses mother to child viral transmission, the knowledge of drug safety linked to fetal neurodevelopment remains an area of active investigation. For example, few studies have suggested that ARV use can be associated with neural tube defects (NTDs) and most notably with the integrase strand transfer inhibitor (INSTI) dolutegravir (DTG). After risk benefit assessments, the World Health Organization (WHO) made recommendations for DTG usage as a first and second-line preferred treatment for infected populations including pregnant women and those of childbearing age. Nonetheless, long-term safety concerns remain for fetal health. This has led to a number of recent studies underscoring the need for biomarkers to elucidate potential mechanisms underlying long-term neurodevelopmental adverse events. With this goal in mind, we now report the inhibition of matrix metalloproteinases (MMPs) activities by INSTIs as an ARV class effect. Balanced MMPs activities play a crucial role in fetal neurodevelopment. Inhibition of MMPs activities by INSTIs during neurodevelopment could be a potential mechanism for adverse events. Thus, comprehensive molecular docking testing of the INSTIs, DTG, bictegravir (BIC), and cabotegravir (CAB), against twenty-three human MMPs showed broad-spectrum inhibition. With a metal chelating chemical property, each of the INSTI were shown to bind Zn++ at the MMP's catalytic domain leading to MMP inhibition but to variable binding energies. These results were validated in myeloid cell culture experiments demonstrating MMP-2 and 9 inhibitions by DTG, BIC and CAB and even at higher degree than doxycycline (DOX). Altogether, these data provide a potential mechanism for how INSTIs could affect fetal neurodevelopment.
ABSTRACT
Colorectal cancer (CRC) is a leading cause of the cancer-related deaths worldwide. Thus, developing novel and targeted therapies for inhibiting CRC progression and metastasis is urgent. Several studies, including ours, have reported a causal role for an upregulated claudin-1 expression in promoting CRC metastasis through the activation of the Src and ß-catenin-signaling. In murine models of colon tumorigenesis, claudin-1 overexpression promotes oncogenic properties such as transformation and invasiveness. Conversely, the downregulation of claudin-1 inhibits colon tumorigenesis. Despite being a desirable target for cancer treatment, there are currently no known claudin-1 inhibitors with antitumor efficacy. Using a rigorous analytical design and implementing in- vitro and in-vivo testing and a brief medicinal chemistry campaign, we identified a claudin-1-specific inhibitor and named it I-6. Despite its high potency, I-6 was rapidly cleared in human liver microsomes. We, therefore, synthesized I-6 analogs and discovered a novel small molecule, PDS-0330. We determined that PDS0330 inhibits claudin-1-dependent CRC progression without exhibiting toxicity in in-vitro and in-vivo models of CRC and that it binds directly and specifically to claudin-1 with micromolar affinity. Further analyses revealed that PDS-0330 exhibits antitumor and chemosensitizer activities with favorable pharmacokinetic properties by inhibiting the association with metastatic oncogene Src. Overall, our data propose that PDS-0330 interferes with claudin-1/Src association to inhibit CRC progression and metastasis. Our findings are of direct clinical relevance and may open new therapeutic opportunities in colon cancer treatment and/or management by targeting claudin-1.
Subject(s)
Colonic Neoplasms , Colorectal Neoplasms , Mice , Humans , Animals , Claudin-1/metabolism , Colonic Neoplasms/pathology , Cell Transformation, Neoplastic/genetics , Carcinogenesis/metabolism , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Cell Line, TumorABSTRACT
The incidence of sudden cardiac death (SCD) in people living with HIV infection (PLWH), especially those with inadequate viral suppression, is high and the reasons for this remain incompletely characterized. The timely opening and closing of type 2 ryanodine receptor (RyR2) is critical for ensuring rhythmic cardiac contraction-relaxation cycles, and the disruption of these processes can elicit Ca2+ waves, ventricular arrhythmias, and SCD. Herein, we show that the HIV protein Tat (HIV-Tat: 0-52 ng/mL) and therapeutic levels of the antiretroviral drugs atazanavir (ATV: 0-25,344 ng/mL), efavirenz (EFV: 0-11,376 ng/mL), and ritonavir (RTV: 0-25,956 ng/mL) bind to and modulate the opening and closing of RyR2. Abacavir (0-14,315 ng/mL), bictegravir (0-22,469 ng/mL), Rilpivirine (0-14,360 ng/mL), and tenofovir disoproxil fumarate (0-18,321 ng/mL) did not alter [3H]ryanodine binding to RyR2. Pretreating RyR2 with low HIV-Tat (14 ng/mL) potentiated the abilities of ATV and RTV to bind to open RyR2 and enhanced their ability to bind to EFV to close RyR2. In silico molecular docking using a Schrodinger Prime protein-protein docking algorithm identified three thermodynamically favored interacting sites for HIV-Tat on RyR2. The most favored site resides between amino acids (AA) 1702-1963; the second favored site resides between AA 467-1465, and the third site resides between AA 201-1816. Collectively, these new data show that HIV-Tat, ATV, EFV, and RTV can bind to and modulate the activity of RyR2 and that HIV-Tat can exacerbate the actions of ATV, EFV, and RTV on RyR2. Whether the modulation of RyR2 by these agents increases the risk of arrhythmias and SCD remains to be explored.
Subject(s)
Anti-HIV Agents , HIV Infections , Humans , Atazanavir Sulfate/pharmacology , Atazanavir Sulfate/therapeutic use , Ritonavir/pharmacology , Ritonavir/therapeutic use , Ryanodine Receptor Calcium Release Channel , HIV Infections/drug therapy , Anti-HIV Agents/adverse effects , Molecular Docking Simulation , Oligopeptides/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic useABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a pandemic of unprecedented scale. This coronavirus enters cells by the interaction of the receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 receptor (hACE2). In this study, we employed a rational structure-based design to propose 22-mer stapled peptides using the structure of the hACE2 α1 helix as a template. These peptides were designed to retain the α-helical character of the natural structure, to enhance binding affinity, and to display a better solubility profile compared to other designed peptides available in the literature. We employed different docking strategies (PATCHDOCK and ZDOCK) followed by a double-step refinement process (FIBERDOCK) to rank our peptides, followed by stability analysis/evaluation of the interaction profile of the best docking predictions using a 500 ns molecular dynamics (MD) simulation, and a further binding affinity analysis by molecular mechanics with generalized Born and surface area (MM/GBSA) method. Our most promising stapled peptides presented a stable profile and could retain important interactions with the RBD in the presence of the E484K RBD mutation. We predict that these peptides can bind to the viral RBD with similar potency to the control NYBSP-4 (a 30-mer experimentally proven peptide inhibitor). Furthermore, our study provides valuable information for the rational design of double-stapled peptide as inhibitors of SARS-CoV-2 infection.
Subject(s)
SARS-CoV-2 , Spike Glycoprotein, Coronavirus/chemistry , Angiotensin-Converting Enzyme 2/chemistry , Antiviral Agents/pharmacology , Binding Sites , COVID-19 , Humans , Molecular Dynamics Simulation , Peptides/pharmacology , Protein Binding , SARS-CoV-2/drug effectsABSTRACT
Inhibitor of nuclear factor kappa-B kinase subunit beta (IKKß) is a key regulator of the cannonical NF-κB pathway. IKKß has been validated as a drug target for pathological conditions, which include chronic inflammatory diseases and cancer. Pharmacological studies revealed that chronic administration of ATP-competitive IKKß inhibitors resulted in unexpected toxicity. We previously reported the discovery of 13-197 as a non-toxic IKKß inhibitor that reduced tumor growth. Here, we show that 13-197 inhibits IKKß in a ATP non-competitive manner and an allosteric pocket at the interface of the kinase and ubiquitin like domains was identified as the potential binding site.
Subject(s)
Adenosine Triphosphate/metabolism , I-kappa B Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Binding Sites/drug effects , Dose-Response Relationship, Drug , Humans , I-kappa B Kinase/metabolism , Molecular Structure , Protein Kinase Inhibitors/chemistry , Small Molecule Libraries/chemistryABSTRACT
Filoviruses, mainly consisting of Ebola viruses (EBOV) and Marburg viruses (MARV), are enveloped negative-strand RNA viruses which can infect humans to cause severe hemorrhagic fevers and outbreaks with high mortality rates. The filovirus infection is mediated by the interaction of viral envelope glycoprotein (GP) and the human endosomal receptor Niemann-Pick C1 (NPC1). Blocking this interaction will prevent the infection. Therefore, we utilized an In silico screening approach to conduct virtual compound screening against the NPC1 receptor-binding site (RBS). Twenty-six top-hit compounds were purchased and evaluated by in vitro cell based inhibition assays against pseudotyped or replication-competent filoviruses. Two classes (A and U) of compounds were identified to have potent inhibitory activity against both Ebola and Marburg viruses. The IC50 values are in the lower level of micromolar concentrations. One compound (compd-A) was found to have a sub-micromolar IC50 value (0.86 µM) against pseudotyped Marburg virus. The cytotoxicity assay (MTT) indicates that compd-A has a moderate cytotoxicity level but the compd-U has much less toxicity and the CC50 value was about 100 µM. Structure-activity relationship (SAR) study has found some analogs of compd-A and -U have reduced the toxicity and enhanced the inhibitory activity. In conclusion, this work has identified several qualified lead-compounds for further drug development against filovirus infection.
Subject(s)
Antiviral Agents/pharmacology , Ebolavirus/drug effects , Filoviridae Infections/virology , Marburgvirus/drug effects , Niemann-Pick C1 Protein/metabolism , Viral Envelope Proteins/metabolism , Virus Internalization/drug effects , Antiviral Agents/chemistry , Binding Sites , Cell Survival , Drug Discovery , Ebolavirus/physiology , Filoviridae Infections/drug therapy , HeLa Cells , Host Microbial Interactions/drug effects , Humans , Inhibitory Concentration 50 , Marburgvirus/physiology , Molecular Docking Simulation , Niemann-Pick C1 Protein/chemistry , Protein Binding , Receptors, Virus/chemistry , Receptors, Virus/metabolismSubject(s)
Angiotensin-Converting Enzyme 2/metabolism , Bromelains/pharmacology , COVID-19 Drug Treatment , COVID-19 , SARS-CoV-2/metabolism , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Animals , COVID-19/metabolism , COVID-19/pathology , Chlorocebus aethiops , Humans , Vero CellsABSTRACT
More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions.
Subject(s)
14-3-3 Proteins/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/drug therapy , Skin Neoplasms/drug therapy , cdc25 Phosphatases/metabolism , 14-3-3 Proteins/genetics , 14-3-3 Proteins/metabolism , 9,10-Dimethyl-1,2-benzanthracene/administration & dosage , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Carcinogens/administration & dosage , Carcinogens/toxicity , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cytoplasm/drug effects , Cytoplasm/metabolism , Female , Humans , Keratinocytes , Male , Mice , Mice, Knockout , Neoplasms, Experimental/chemically induced , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Protein Multimerization/drug effects , Skin Neoplasms/pathology , Tetradecanoylphorbol Acetate/administration & dosage , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/toxicity , Xenograft Model Antitumor AssaysABSTRACT
EP67 is a second-generation, human C5a-derived decapeptide agonist of C5a receptor 1 (C5aR1/CD88) that selectively activates mononuclear phagocytes over neutrophils to potentiate protective innate and adaptive immune responses while potentially minimizing neutrophil-mediated toxicity. Pro7 and N-methyl-Leu8 (Me-Leu8) amino acid residues within EP67 likely induce backbone structural changes that increase potency and selective activation of mononuclear phagocytes over neutrophils versus first-generation EP54. The low coupling efficiency between Pro7 and Me-Leu8 and challenging purification by HPLC, however, greatly increase scale-up costs of EP67 for clinical use. Thus, the goal of this study was to determine whether replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes (cyclohexylalanine7 and/or leucine8) sufficiently preserves EP67 activity in primary human mononuclear phagocytes and neutrophils. We found that EP67 analogues had similar potency, efficacy, and selective activation of mononuclear phagocytes over neutrophils. Thus, replacing Pro7 and/or Me-Leu8 with large-scale amenable amino acid residues predicted to induce similar structural changes is a suitable strategy to overcome scale-up challenges with EP67.
Subject(s)
Adjuvants, Immunologic/chemistry , Complement C5a , Oligopeptides/chemistry , Amino Acid Substitution , HumansABSTRACT
Fruit flies must compensate for the limited light gathered by the tiny facets of their eyes, and image motion during flight lowers light catch even further. Motion blur is especially problematic in fast regions of the visual field, perpendicular to forward motion, but flow fields also contain slower regions, less affected by blur. To test whether fruit flies shift their attention to predictably slower regions of a flow field, we placed flies in an arena simulating forward flight and measured responses to turning cues in different visual areas. We find that during fast forward flight, fruit flies respond more strongly to turning cues presented directly in front, and less strongly to cues presented to the sides, supporting the hypothesis that flying fruit flies shift visual attention to slower moving regions less affected by motion blur.
Subject(s)
Optic Flow , Animals , Attention , Drosophila melanogaster , Flight, Animal , Visual FieldsABSTRACT
Zika virus (ZIKV), an emerging arbovirus, has become a major human health concern globally due to its association with congenital abnormalities and neurological diseases. Licensed vaccines or antivirals against ZIKV are currently unavailable. Here, by employing a structure-based approach targeting the ZIKV RNA-dependent RNA polymerase (RdRp), we conducted in silico screening of a library of 100,000 small molecules and tested the top ten lead compounds for their ability to inhibit the virus replication in cell-based in vitro assays. One compound, 3-chloro-N-[({4-[4-(2-thienylcarbonyl)-1-piperazinyl]phenyl}amino)carbonothioyl]-1-benzothiophene-2-carboxamide (TPB), potently inhibited ZIKV replication at sub-micromolar concentrations. Molecular docking analysis suggests that TPB binds to the catalytic active site of the RdRp and therefore likely blocks the viral RNA synthesis by an allosteric effect. The IC50 and the CC50 of TPB in Vero cells were 94 nM and 19.4 µM, respectively, yielding a high selective index of 206. In in vivo studies using immunocompetent mice, TPB reduced ZIKV viremia significantly, indicating TPB as a potential drug candidate for ZIKV infections.
Subject(s)
Antiviral Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Virus Replication/drug effects , Zika Virus/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Cell Survival , Chlorocebus aethiops , Computer Simulation , Inhibitory Concentration 50 , Mice, Inbred BALB C , Molecular Docking Simulation , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Vero Cells , Viral Load/drug effects , Zika Virus/enzymology , Zika Virus/physiology , Zika Virus Infection/drug therapy , Zika Virus Infection/virologyABSTRACT
The bacterial protein DnaK promotes folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. Assisted refolding is especially important under stress conditions induced by antibiotic therapies reducing the desired bactericidal effects. DnaK is supposedly targeted by proline-rich antimicrobial peptides (PrAMPs), but Escherichia coli ΔdnaK mutants and wild type strains are equally susceptible indicating further intracellular targets, such as the 70S ribosome. Crystal structures of PrAMPDnaK- complexes revealed forward and reverse binding modes at the substrate binding domain. Here, we used these ligand-target structures for the first time to rationally optimize peptides using molecular modeling and docking leading to the prediction of four-residue long sequences for improved binding to DnaK. When these sequences were used to replace the original sequence stretch in Onc72, most peptides showed significantly reduced dissociation constants (Kd) determined by fluorescence polarization. In a second approach, the X-ray structures of Api88 and Onc72 bound to DnaK were examined to predict substitutions prone to stronger interactions. Among the 36 peptides obtained from both approaches, six derivatives bound to DnaK with more than 10-fold higher affinities (Kd values in the low micromolar to nanomolar range). Peptides binding stronger to DnaK showed the same minimal inhibitory concentrations against wild type E. coli as the original peptide, but were slightly less active for ΔdnaK mutants. However, one peptide was able to overcome the resistance in an E. coli mutant lacking the SbmA transporter obligatory for the uptake of PrAMPs including Api88 and Onc72. Thus, it´s tempting to speculate that DnaK might be involved in the translocation of PrAMPs into E. coli.
Subject(s)
Anti-Bacterial Agents/metabolism , Antimicrobial Cationic Peptides/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/drug effects , Escherichia coli/metabolism , HSP70 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Antimicrobial Cationic Peptides/chemistry , Binding Sites , Escherichia coli/genetics , Escherichia coli Proteins/genetics , HSP70 Heat-Shock Proteins/genetics , Klebsiella pneumoniae/drug effects , Membrane Transport Proteins/genetics , Molecular Docking Simulation , Protein Binding , Protein Refolding , Protein Structure, Tertiary , Protein TransportABSTRACT
One factor that influences estimates of time since death using entomological evidence is whether or not blow flies nocturnally oviposit. Field studies focusing on egg laying have found it occurs on an inconsistent basis. A key but poorly understood factor in nocturnal oviposition is a blow fly's ability to locate carrion under low light levels. It has been speculated that blow flies are more likely to walk than fly to carrion during the night, but this has not been empirically tested. We directly compared guided walking versus flying using infrared sensors under low light levels in laboratory conditions for Chrysomya megacephala (F.) (Diptera: Calliphoridae), a blow fly previously described to be nocturnal. We found C. megacephala is more likely to walk than fly toward carrion under low light levels (p=0.016). We did not, however, find differences between males and females for walking (p=0.48) or flying (p=0.42) despite male C. megacephala possessing eyes better suited for increased light capture. These results demonstrate the need to better understand where blow flies go at night, as bodies found within a fly's walking distance are more likely to be colonized.
Subject(s)
Diptera/physiology , Light , Walking/physiology , Animals , Female , Flight, Animal , Male , OvipositionABSTRACT
Yes-associated protein (YAP) is an effector of the Hippo tumor suppressor pathway. The functional significance of YAP in prostate cancer has remained elusive. In this study, we first show that enhanced expression of YAP is able to transform immortalized prostate epithelial cells and promote migration and invasion in both immortalized and cancerous prostate cells. We found that YAP mRNA was upregulated in androgen-insensitive prostate cancer cells (LNCaP-C81 and LNCaP-C4-2 cells) compared to the level in androgen-sensitive LNCaP cells. Importantly, ectopic expression of YAP activated androgen receptor signaling and was sufficient to promote LNCaP cells from an androgen-sensitive state to an androgen-insensitive state in vitro, and YAP conferred castration resistance in vivo. Accordingly, YAP knockdown greatly reduced the rates of migration and invasion of LNCaP-C4-2 cells and under androgen deprivation conditions largely blocked cell division in LNCaP-C4-2 cells. Mechanistically, we found that extracellular signal-regulated kinase-ribosomal s6 kinase signaling was downstream of YAP for cell survival, migration, and invasion in androgen-insensitive cells. Finally, immunohistochemistry showed significant upregulation and hyperactivation of YAP in castration-resistant prostate tumors compared to their levels in hormone-responsive prostate tumors. Together, our results identify YAP to be a novel regulator in prostate cancer cell motility, invasion, and castration-resistant growth and as a potential therapeutic target for metastatic castration-resistant prostate cancer (CRPC).
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Gene Expression Regulation, Neoplastic , Phosphoproteins/metabolism , Prostate/pathology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Signal Transduction , Adaptor Proteins, Signal Transducing/genetics , Androgens/metabolism , Animals , Castration , Cell Movement , Cell Proliferation , Gene Knockdown Techniques , HEK293 Cells , Hippo Signaling Pathway , Humans , MAP Kinase Signaling System , Male , Mice, SCID , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Phosphoproteins/genetics , Prostate/metabolism , Prostatic Neoplasms/genetics , Protein Serine-Threonine Kinases/metabolism , Transcription Factors , Up-Regulation , YAP-Signaling ProteinsABSTRACT
Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell-cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant "activated PTK-selective" ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.
Subject(s)
Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-cbl/physiology , Ubiquitination/physiology , Amino Acid Sequence , Animals , Humans , Mice , Models, Biological , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , Ubiquitin/metabolismABSTRACT
Colorectal cancers with metastatic potential secrete the glycoprotein carcinoembryonic antigen (CEA). CEA has been implicated in colorectal cancer metastasis by inducing Kupffer cells to produce inflammatory cytokines which, in turn, make the hepatic micro-environment ideal for tumor cell implantation. CEA binds to the heterogeneous ribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells. The amino acid sequence in CEA, which binds the hnRNP M receptor, is Tyr-Pro-Glu-Leu-Pro-Lys. In this study, the structure of Ac-Tyr-Pro-Glu-Leu-Pro-Lys-NH2 (YPELPK) was investigated using electronic circular dichroism, vibrational circular dichroism, and molecular dynamics simulations. The binding of the peptide to hnRNP M was also investigated using molecular docking calculations. The biological activity of YPELPK was studied using differentiated human THP-1 cells, which express hnRNP M on their surface and secrete IL-6 when stimulated by CEA. YPELPK forms a stable polyproline-II helix and stimulates IL-6 production of THP-1 cells at micromolar concentrations.
Subject(s)
Carcinoembryonic Antigen/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group M/agonists , Peptide Fragments/chemistry , Amino Acid Sequence , Amino Acid Substitution , Carcinoembryonic Antigen/pharmacology , Cell Line , Heterogeneous-Nuclear Ribonucleoprotein Group M/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group M/metabolism , Humans , Hydrogen Bonding , Interleukin-6/biosynthesis , Molecular Dynamics Simulation , Peptide Fragments/pharmacology , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Surface Properties , ThermodynamicsABSTRACT
We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape. Our results suggest that the pentapeptide is an ideal starting point for the development of inhibitors against Cbl driven diseases.
Subject(s)
Models, Molecular , Oligopeptides/chemistry , Oncogene Protein v-cbl/chemistry , Protein-Tyrosine Kinases/chemistry , Binding Sites , Oncogene Protein v-cbl/metabolism , Peptide Library , Protein Binding , Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , ThermodynamicsABSTRACT
Breast cancer gene 1 carboxy terminus (BRCT) domains are found in a number of proteins that are important for DNA damage response (DDR). The BRCT domains bind phosphorylated proteins and these protein-protein interactions are essential for DDR and DNA repair. High affinity domain specific inhibitors are needed to facilitate the dissection of the protein-protein interactions in the DDR signaling. The BRCT domains of BRCA1 bind phosphorylated protein through a pSXXF consensus recognition motif. We identified a hydrophobic pocket at the P-1 position of the pSXXF binding site. Here we conducted a structure-guided synthesis of peptide analogs with hydrophobic functional groups at the P-1 position. Evaluation of these led to the identification of a peptide mimic 15 with a inhibitory constant (K(i)) of 40 nM for BRCT(BRCA1). Analysis of the TopBP1 and MDC1 BRCT domains suggests a similar approach is viable to design high affinity inhibitors.
ABSTRACT
Nuclear Factor κ B is implicated in tumor progression and chronic inflammatory diseases and is regulated by IκB kinase ß (IKKß). The crystal structure of IKKß has been recently solved for Xenopus laevis. Homology models of human IKKß have been developed prior to and after the crystal structure was solved. Here, we compare four models of human IKKß and evaluate their performance in both broad and focused library docking studies.
Subject(s)
I-kappa B Kinase/chemistry , Animals , Binding Sites , Humans , Models, Molecular , Protein Conformation , Structural Homology, ProteinABSTRACT
Misfolding and aggregation of amyloid ß-40 (Aß-40) peptide play key roles in the development of Alzheimer's disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aß-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aß-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.