Inhibition Of Complex Formation Research Articles

Binding Mechanism between Platelet Glycoprotein and Cyclic Peptide Elucidated by McMD-Based Dynamic Docking.

The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the platelet glycoprotein Ibα (GPIbα) and the von Willebrand factor (vWF) by forming a complex with GPIbα. To study the binding mechanism between GPIbα and OS1 and, therefore, the inhibition mechanism of the protein-protein GPIbα-vWF complex, we have applied our multicanonical molecular dynamics (McMD)-based dynamic docking protocol starting from the unbound state of the peptide. Our simulations have reproduced the experimental complex structure, although the top-ranking structure was an intermediary one, where the peptide was bound in the same location as in the experimental structure; however, the β-switch of GPIbα attained a different conformation. Our analysis showed that subsequent refolding of the β-switch results in a more stable binding configuration, although the transition to the native configuration appears to take some time, during which OS1 could dissociate. Our results show that conformational changes in the β-switch are crucial for successful binding of OS1. Furthermore, we identified several allosteric binding sites of GPIbα that might also interfere with vWF binding, and optimization of the peptide to target these allosteric sites might lead to a more effective inhibitor, as these are not dependent on the β-switch conformation.

A Stapled Peptide Inhibitor Targeting the Binding Interface of N6‐Adenosine‐Methyltransferase Subunits METTL3 and METTL14 for Cancer Therapy

AbstractMETTL3, a primary methyltransferase catalyzing the RNA N6‐methyladenosine (m6A) modification, has been identified as an oncogene in several cancer types and thus nominated as a potentially effective target for therapeutic inhibition. However, current options using this strategy are limited. In this study, we targeted protein–protein interactions at the METTL3–METTL14 binding interface to inhibit complex formation and subsequent catalysis of the RNA m6A modification. Among candidate peptides, RM3 exhibited the highest anti‐cancer potency, inhibiting METTL3 activity while also facilitating its proteasomal degradation. We then designed a stapled peptide inhibitor (RSM3) with enhanced peptide stability and formation of the α‐helical secondary structure required for METTL3 interaction. Functional and transcriptomic analysis in vivo indicated that RSM3 induced upregulation of programmed cell death‐related genes while inhibiting cancer‐promoting signals. Furthermore, tumor growth was significantly suppressed while apoptosis was enhanced upon RSM3 treatment, accompanied by increased METTL3 degradation, and reduced global RNA methylation levels in two in vivo tumor models. This peptide inhibitor thus exploits a mechanism distinct from other small‐molecule competitive inhibitors to inhibit oncogenic METTL3 activity. Our findings collectively highlight the potential of targeting METTL3 in cancer therapies through peptide‐based inhibition of complex formation and proteolytic degradation.

A Stapled Peptide Inhibitor Targeting the Binding Interface of N6-Adenosine-Methyltransferase Subunits METTL3 and METTL14 for Cancer Therapy.

METTL3, a primary methyltransferase catalyzing the RNA N6-methyladenosine (m6A) modification, has been identified as an oncogene in several cancer types and thus nominated as a potentially effective target for therapeutic inhibition. However, current options using this strategy are limited. In this study, we targeted protein-protein interactions at the METTL3-METTL14 binding interface to inhibit complex formation and subsequent catalysis of the RNA m6A modification. Among candidate peptides, RM3 exhibited the highest anti-cancer potency, inhibiting METTL3 activity while also facilitating its proteasomal degradation. We then designed a stapled peptide inhibitor (RSM3) with enhanced peptide stability and formation of the α-helical secondary structure required for METTL3 interaction. Functional and transcriptomic analysis in vivo indicated that RSM3 induced upregulation of programmed cell death-related genes while inhibiting cancer-promoting signals. Furthermore, tumor growth was significantly suppressed while apoptosis was enhanced upon RSM3 treatment, accompanied by increased METTL3 degradation, and reduced global RNA methylation levels in two in vivo tumor models. This peptide inhibitor thus exploits a mechanism distinct from other small-molecule competitive inhibitors to inhibit oncogenic METTL3 activity. Our findings collectively highlight the potential of targeting METTL3 in cancer therapies through peptide-based inhibition of complex formation and proteolytic degradation.

Effect of pre-treatments and frying conditions on the formation of starch-lipid complex in potato starch chips during deep-frying process

This study examined the influence of various pretreatment methods, frying durations, and temperatures, as well as the type of frying oil, on the formation and structure of starch-lipid complexes in fried potato chips. Potato starch was processed into dough, sliced, and subjected to deep frying following various pretreatments. Structural analysis showed that steaming as a pretreatment facilitated the generation of V-type starch-lipid complexes, whereas resistant starch type III (RS3) materialized in the desiccated samples instead of the anticipated complexes. The rate of starch-lipid complex formation initially surged but subsequently declined as treatment time increased. A reduction in treatment temperature from 190 °C to 170 °C was conducive to complex formation. Moreover, the maximum relative crystallinity (19.74 %) and ΔH value (7.76 J/g) were recorded for potato starch slices pretreated by steaming and frying in palm oil. Rapeseed oil, which is rich in unsaturated fatty acids (89.98 %), inhibits complex formation. The study concludes that pretreatment methods exert a substantial effect on the formation of starch-lipid complexes and that extended frying duration and elevated temperature may reduce this formation. Oils with longer-chain fatty acids and a lower degree of unsaturation were favorable for complex formation.

Formation and clearance of TNF-TNF inhibitor complexes during TNF inhibitor treatment.

Millions of patients with inflammatory diseases are treated with tumour necrosis factor (TNF) inhibitors (TNFi). Individual treatment response varies, in part related to variable drug clearance. The role of TNF-TNFi complexes in clearance of the different TNFi is controversial. Moreover, mechanistic insight into the structural aspects and biological significance of TNF-TNFi complexes is lacking. We hypothesized a role for Fc-mediated clearance of TNF-TNFi immune complexes. Therefore, we investigated circulating TNF-TNFi complexes upon treatment with certolizumab-lacking Fc tails-in comparison with adalimumab, golimumab, infliximab and etanercept. Drug-tolerant ELISAs were developed and used to quantify TNF during adalimumab, golimumab, etanercept, certolizumab and infliximab treatment in patients with inflammatory arthritis or ulcerative colitis for a maximum follow-up of 1 year. Effects on in vitro TNF production and Fc-mediated uptake of TNF-TNFi complexes were investigated for all five TNFi. Circulating TNF concentrations were >20-fold higher during certolizumab treatment compared with adalimumab, reaching up to 23.1 ng·ml-1 . Internalization of TNF-TNFi complexes by macrophages depended on Fc valency, with efficient uptake for the full antibody TNFi (three Fc tails), but little or no uptake for etanercept and certolizumab (one and zero Fc tail, respectively). TNF production was not affected by TNFi. Total TNF load did not affect clearance rate of total TNFi. Differences in TNFi structure profoundly affect clearance of TNF, while it is unlikely that TNF itself significantly contributes to target-mediated drug disposition of TNFi.

Abstract 12953: Targeting NEDD9 and SMAD3 in the Pathogenesis of Chronic Thromboembolic Pulmonary Hypertension

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by unresolved pulmonary thrombi due, in part, to impaired fibrinolysis. We showed previously that oxidation of the Cas protein NEDD9 at cysteine-18 (Cys 18 ) inhibits complex formation between NEDD9 and the anti-fibrinolytic transcription factor SMAD3 in human pulmonary artery endothelial cells (HPAECs). However, the effect of NEDD9-Cys 18 oxidation on SMAD3 bioactivity in HPAECs is not known. Hypothesis: NEDD9-Cys 18 oxidation increases bioavailable SMAD3, which upregulates SERPINE1 that encodes plasminogen activator inhibitor (PAI)-1 to inhibit fibrinolysis pathways in HPAECs. Methods/Results: To assess the effect of SMAD3 on SERPINE1 transcription, HPAECs were transfected with cDNA-SMAD3 (1 μg/μl) for 72 hr to induce SMAD3 overexpression. Compared to vehicle (V) control, cDNA-SMAD3 increased PAI-1 and SERPINE1 expression by 35% (P=0.004) by immunoblot and 70% (P=0.003) by real-time PCR, respectively (N=3). To determine the effect of NEDD9 inhibition on SMAD3 activation, we analyzed the ratio of P-SMAD-Ser423/425:SMAD3 by immunoblot. Compared to scrambled RNA (scRNA), transfection with siRNA against NEDD9 (siNEDD9) for 48 hr increased the relative quantity of activated SMAD3 by 1.4-fold (P=0.006) and upregulated PAI-1 (P=0.004) expression by 1.7-fold in HPAECs (N=4). Next, we monitored the effect of oxidant stress on SMAD3 bioactivity. Compared to V-control, the expression of NEDD9, total SMAD3, and P-SMAD3 was increased by 66% (P=0.04), 81% (P=0.03), and 29% (P=0.04), respectively, in HPAECs treated with hydrogen peroxide (H 2 O 2 ) (500 μM) for 20 min. Next, the effect of NEDD9 silencing on H 2 O 2 accumulation in HPAECs was measured by Amplex Red assay. HPAECs were transfected with scRNA or siNEDD9 for 48 hr followed by measurement of H 2 O 2 production. Compared to scRNA, NEDD9 silencing significantly decreased H 2 O 2 concentration by 56% (N=4, P=0.02). Conclusion: These data demonstrate that oxidant stress increases SMAD3 activity and SMAD3-dependent upregulation of PAI-1 through a mechanism involving redox regulation of NEDD9. Identifying prothrombotic control of SMAD3 by NEDD9 may have important therapeutic implications for CTEPH.

In silico identification of modulators of J domain protein-Hsp70 interactions in Plasmodium falciparum: a drug repurposing strategy against malaria.

Plasmodium falciparum is a unicellular, intracellular protozoan parasite, and the causative agent of malaria in humans, a deadly vector borne infectious disease. A key phase of malaria pathology, is the invasion of human erythrocytes, resulting in drastic remodeling by exported parasite proteins, including molecular chaperones and co-chaperones. The survival of the parasite within the human host is mediated by P. falciparum heat shock protein 70s (PfHsp70s) and J domain proteins (PfJDPs), functioning as chaperones-co-chaperones partnerships. Two complexes have been shown to be important for survival and pathology of the malaria parasite: PfHsp70-x-PFE0055c (exported); and PfHsp70-2-PfSec63 (endoplasmic reticulum). Virtual screening was conducted on the drug repurposing library, the Pandemic Response Box, to identify small-molecules that could specifically disrupt these chaperone complexes. Five top ranked compounds possessing preferential binding affinity for the malarial chaperone system compared to the human system, were identified; three top PfHsp70-PfJDP binders, MBX 1641, zoliflodacin and itraconazole; and two top J domain binders, ezetimibe and a benzo-diazepinone. These compounds were validated by repeat molecular dockings and molecular dynamics simulation, resulting in all the compounds, except for MBX 1461, being confirmed to bind preferentially to the malarial chaperone system. A detailed contact analysis of the PfHsp70-PfJDP binders identified two different types of modulators, those that potentially inhibit complex formation (MBX 1461), and those that potentially stabilize the complex (zoliflodacin and itraconazole). These data suggested that zoliflodacin and itraconazole are potential novel modulators specific to the malarial system. A detailed contact analysis of the J domain binders (ezetimibe and the benzo-diazepinone), revealed that they bound with not only greater affinity but also a better pose to the malarial J domain compared to that of the human system. These data suggested that ezetimibe and the benzo-diazepinone are potential specific inhibitors of the malarial chaperone system. Both itraconazole and ezetimibe are FDA-approved drugs, possess anti-malarial activity and have recently been repurposed for the treatment of cancer. This is the first time that such drug-like compounds have been identified as potential modulators of PfHsp70-PfJDP complexes, and they represent novel candidates for validation and development into anti-malarial drugs.

Abstract 1824: Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity

Abstract Interleukin-18 (IL18) is a proinflammatory cytokine that modulates both innate and adaptive immune responses. Although recombinant mIL18 has demonstrated efficacy in mouse tumor models, recombinant human IL18 has not been efficacious in clinical studies, likely due to upregulation of IL18 binding protein (IL18BP), a negative regulator of the IL18 signaling axis. IL18BP is induced 10 - 100 fold after IL18 administration, binds to IL18 with high affinity, and prevents IL18 binding to the IL18 receptor. Analysis of expression data from the TCGA database as well as archived patient samples from select indications revealed that both IL18 and IL18BP are co-expressed in a number of tumor types, suggesting there may be a preexisting pool of IL18/IL18BP complex at the site of the tumor in some patients. Inhibition of IL18/IL18BP binding using a monoclonal antibody may therefore allow release of IL18 in situ, leading to IL18-mediated proinflammatory effects and enhanced anti-tumor immunity. Although the interaction of IL18 with IL18BP is a high affinity interaction, we have been able to identify very high affinity antibodies which can both inhibit complex formation and disrupt existing mouse IL18/IL18BP complexes. These antibodies are active in a number of cell-based functional assays, resulting in potent stimulation of interferon gamma production from cell lines or splenocytes. In vivo, we observed efficacy in a mouse syngeneic tumor model using an anti-mIL18BP antibody. RNA and protein analysis demonstrated expression of both IL18BP and IL18 within the tumors. Combined, these data suggest that inhibition of IL18/IL18BP binding may prove efficacious in the treatment of cancers where existing pools of IL18/IL18BP complexes prevent an IL18-mediated proinflammatory response. Experiments exploring anti-IL18BP therapy in combination with other checkpoint inhibitors such as anti-PD-1 are under current investigation. Citation Format: H. Toni Jun, Deborah A. Witherden, Eric K. Elliot, Christine M. Chidester, Maggie Willen, Bryan R. Peguero, Merrick Chai, Robert A. Horlick, David J. King. Discovery and evaluation of an anti-IL18BP antibody to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1824.

Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins.

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex

In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Herein, we performed extensive molecular dynamics simulations (totally ∼13 μs) on both heterodimer and heterotrimer to elucidate at an atomic level how each p53-degradation-defective HPV16 E6 mutant reduces the structural stabilities of the two complexes. Our simulations reveal that the three E6 mutations destabilize the structure of E6/E6AP/p53 complex through distinct mechanisms. Although F47RE6 mutation has no effect on the structure of E6/E6AP heterodimer, it results in an electrostatic repulsion between R47E6 and R290p53, which is unfavorable for E6-p53 binding. R102AE6 mutation destabilizes the structure of E6/E6AP heterodimer and significantly disrupts hydrophobic and cation-π interactions between F47E6 and E286p53/L298p53/R290p53. L50EE6 mutation impairs both E6 interdomain interactions (especially F47-K108 cation-π interaction) and E6-E6AP intermolecular interactions important for the stabilization of E6/E6AP heterodimer. This study identifies the intra- and intermolecular interactions crucial for the complex stability, which may provide mechanistic insights into the inhibition of complex formation by the three HPV16 E6 mutations.

Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD.

The humoral response to the SARS-CoV-2 S protein determines the development of protective immunity against this infection. The standard neutralizing antibodies detection method is a live virus neutralization test. It can be replaced with an ELISA-based surrogate virus neutralization test (sVNT), measuring the ability of serum antibodies to inhibit complex formation between the receptor-binding domain (RBD) of the S protein and the cellular ACE2 receptor. There are conflicting research data on the sVNT methodology and the reliability of its results. We show that the performance of sVNT dramatically improves when the intact RBD from the Wuhan-Hu-1 virus variant is used as the plate coating reagent, and the HRP-conjugated soluble ACE2 is used as the detection reagent. This design omits the pre-incubation step in separate tubes or separate microplate and allows the simple quantification of the results using the linear regression, utilizing only 3–4 test sample dilutions. When this sVNT was performed for 73 convalescent plasma samples, its results showed a very strong correlation with VNT (Spearman’s Rho 0.83). For the RBD, bearing three amino acid substitutions and corresponding to the SARS-CoV-2 beta variant, the inhibitory strength was diminished for 18 out of 20 randomly chosen serum samples, and the magnitude of this decrease was not similar to the change in overall anti-RBD IgG level. The sVNT assay design with the ACE2-HRP is preferable over the assay with the RBD-HRP reagent and is suitable for mass screening of neutralizing antibodies titers.

Tailoring the effect of antithrombin-targeting therapy in haemophilia A using in silico thrombin generation

Factor (F) VIII deficiency causes bleeding in haemophilia A patients because of the reduced formation of procoagulant enzyme thrombin, which is needed to make the blood clot. We measured the dynamics of coagulation in haemophilia A patients by measuring thrombin generation (TG). Additionally, we quantified the procoagulant process of prothrombin conversion and anticoagulant process of thrombin inhibitor complex formation. In haemophilia A, prothrombin conversion is severely reduced, causing TG to be low. Nevertheless, the thrombin inactivation capacity of these patients is comparable to that in healthy subjects, leading to a severe imbalance between procoagulant and anticoagulant processes and a subsequent increased bleeding risk. A novel therapy in haemophilia A is the targeting of anticoagulant pathway, e.g. thrombin inhibitor antithrombin (AT), to restore the haemostatic balance. We simulated the effect of AT reduction on TG in silico. Lowering AT levels restored TG dose-dependently and an AT reduction of 90–95% led to almost normal TG in most patients . However, the variation in response to AT reduction was large between patients, indicating that this approach should be tailored to each individual patients. Ideally, TG and thrombin dynamics simulation could in the future contribute to the management of patients undergoing AT targeting therapy.

14-3-3γ prevents centrosome duplication by inhibiting NPM1 function.

14-3-3 proteins bind to ligands via phospho-serine containing consensus motifs. However, the molecular mechanisms underlying complex formation and dissociation between 14-3-3 proteins and their ligands remain unclear. We identified two conserved acidic residues in the 14-3-3 peptide-binding pocket (D129 and E136) that potentially regulate complex formation and dissociation. Altering these residues to alanine led to opposing effects on centrosome duplication. D129A inhibited centrosome duplication, whereas E136A stimulated centrosome amplification. These results were due to the differing abilities of these mutant proteins to form a complex with NPM1. Inhibiting complex formation between NPM1 and 14-3-3γ led to an increase in centrosome duplication and over-rode the ability of D129A to inhibit centrosome duplication. We identify a novel role of 14-3-3γ in regulating centrosome licensing and a novel mechanism underlying the formation and dissociation of 14-3-3 ligand complexes dictated by conserved residues in the 14-3-3 family.

Naturally acquired blocking human monoclonal antibodies to Plasmodiumvivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

Study of cytostatic and cytotoxic characteristics of modified peptide CDK4/6 inhibitors – functional analogs of p16INK4a (90-97)

Background. Application of mathematical modeling for search for biologically active molecules is currently a promising scientific approach. Application of numerical algorithms for optimization of peptide sequences and molecular dynamics allowed to obtain modified peptide sequences that are functional analogs of the sequence of natural inhibitor of cyclin-dependent kinase 4/6 p16INK4a.The study objective is to establish biological characteristics of peptide sequences of CDK 4/6 inhibitors obtained using mathematical modeling.Materials and methods. The studies were performed in vitro using tumor cell lines (MCF-7, А549, SKOV-3, НСТ116). Apoptosis level, cell distribution per cell cycle stages, changes in Bcl-2 expression, changes in the level of phosphorylated pRb under the effect of the studied molecules were investigated using flow cytometry. Proliferation dynamics of cell populations were studied using RTCA iCELLIgence biosensor technology.Results. Peptide sequences obtained using mathematical modeling decrease the level of phosphorylated pRb, Bcl-2 expression when applied to actively proliferating cells. These proteins serve as molecular targets for the cyclin-dependent kinase 4/6–cyclin D complex, and changes in pRb and Bcl-2 levels might indicate inhibition of complex formation. Consequently, decreased proliferative activity and increased apoptosis were observed. Effectiveness of the peptide sequences depended on their molecular structure and type of the used cell line. Two (2) of the studied modified peptide sequences have higher antiproliferative and proapoptotic effect on tumor cells than native p16INK4a (90-97) sequence.Conclusion. Application of mathematical modeling for search and development of functionally active molecules allowed to create peptide sequences with stronger cyclin-dependent kinase inhibitor effect than p16INK4a, the native inhibitor CDK4/6.

High-Affinity Binding of LDL Receptor-Related Protein 1 to Matrix Metalloprotease 1 Requires Protease:Inhibitor Complex Formation.

Matrix metalloprotease (MMP) activation contributes to the degradation of the extracellular matrix (ECM), resulting in a multitude of pathologies. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted endocytic and signaling receptor that is responsible for internalization and lysosomal degradation of diverse proteases, protease inhibitors, and lipoproteins along with numerous other proteins. In this study, we identified MMP-1 as a novel LRP1 ligand. Binding studies employing surface plasmon resonance revealed that both proMMP-1 and active MMP-1 bind to purified LRP1 with equilibrium dissociation constants (KD) of 19 and 25 nM, respectively. We observed that human aortic smooth muscle cells readily internalize and degrade 125I-labeled proMMP-1 in an LRP1-mediated process. Our binding data also revealed that all tissue inhibitors of metalloproteases (TIMPs) bind to LRP1 with KD values ranging from 23 to 33 nM. Interestingly, the MMP-1/TIMP-1 complex bound to LRP1 with an affinity (KD = 0.6 nM) that was 30-fold higher than that of either component alone, revealing that LRP1 prefers the protease:inhibitor complex as a ligand. Of note, modification of lysine residues on either proMMP-1 or TIMP-1 ablated the ability of the MMP-1/TIMP-1 complex to bind to LRP1. LRP1's preferential binding to enzyme:inhibitor complexes was further supported by the higher binding affinity for proMMP-9/TIMP-1 complexes than for either of these two components alone. LRP1 has four clusters of ligand-binding repeats, and MMP-1, TIMP-1, and MMP-1/TIMP-1 complexes bound to cluster III most avidly. Our results reveal an important role for LRP1 in controlling ECM homeostasis by regulating MMP-1 and MMP-9 levels.

Selective Modulation of the Protease Activated Protein C Using Exosite Inhibiting Aptamers.

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities. Nonanticoagulant APC mutants show beneficial effects as cytoprotective agents. To study, if such biased APC signaling can be achieved by APC-binding ligands, the aptamer technology has been used. A G-quadruplex-containing aptamer, G-NB3, has been selected that binds to the basic exosite of APC with a KD of 0.2 nM and shows no binding to APC-related serine proteases or the zymogen protein C. G-NB3 inhibits the inactivation of activated cofactors V and VIII with IC50 values of 11.6 and 13.1 nM, respectively, without inhibiting the cytoprotective and anti-inflammatory functions of APC as tested using a staurosporine-induced apoptosis assay and a vascular barrier protection assay. In addition, G-NB3 prolongs the plasma half-life of APC through inhibition of APC-serine protease inhibitor complex formation. These physicochemical and functional characteristics qualify G-NB3 as a promising therapeutic agent usable to enhance the cytoprotective functions of APC without increasing the risk of APC-related hemorrhage.

Intramolecular Interactions Dominate the Autoregulation of Escherichia coli Stringent Factor RelA.

Amino acid starvation in Escherichia coli activates the enzymatic activity of the stringent factor RelA, leading to accumulation of the alarmone nucleotide (p)ppGpp. The alarmone acts as an intercellular messenger to regulate transcription, translation and metabolism to mediate bacterial stress adaptation. The enzymatic activity of RelA is subject to multi-layered allosteric control executed both by ligands – such as “starved” ribosomal complexes, deacylated tRNA and pppGpp – and by individual RelA domains. The auto-regulation of RelA is proposed to act either in cis (inhibition of the enzymatic activity of the N-terminal region, NTD, by regulatory C-terminal region, CTD) or in trans (CTD-mediated dimerization leading to enzyme inhibition). In this report, we probed the regulatory roles of the individual domains of E. coli RelA and our results are not indicative of RelA dimerization being the key regulatory mechanism. First, at growth-permitting levels, ectopic expression of RelA CTD does not interfere with activation of native RelA, indicating lack of regulation via inhibitory complex formation in the cell. Second, in our biochemical assays, increasing RelA concentration does not decrease the enzyme activity, as would be expected in the case of efficient auto-inhibition via dimerization. Third, while high-level CTD expression efficiently inhibits the growth, the effect is independent of native RelA and is mediated by direct inhibition of protein synthesis, likely via direct interaction with the ribosomal A-site. Finally, deletion of the RRM domain of the CTD region leads to growth inhibition mediated by accumulation of (p)ppGpp, suggesting de-regulation of the synthetic activity in this mutant.

Specific heme binding to heme regulatory motifs in iron regulatory proteins and its functional significance

Iron regulatory proteins (IRPs) control iron metabolism in mammalian cells by binding to the iron-responsive element (IRE) in the target mRNA. Heme regulatory motifs (HRMs) are conserved in the two IRP homologues IRP1 and IRP2 that specifically bind to two and three heme equivalents, respectively; however, only the heme binding to the iron-dependent degradation (IDD) domain of IRP2 causes heme-mediated oxidation, which does not occur in IRP1. Therefore, the functional significance of conserved HRMs outside the IDD domain is yet unclear. In this study, spectroscopic heme titration with IRP mutants confirmed heme binding to each HRM in IRPs, and the effect of heme binding to HRMs on IRE binding was examined. Native polyacrylamide gel electrophoresis analysis revealed that heme binding to HRMs near the IRE binding site inhibits complex formation between IRPs and IRE without oxidative modification, indicating that the function of HRMs varies outside and within the IDD domain. However, the formation of a typical reactive oxygen species (ROS), hydrogen peroxide, was spectroscopically detected in both heme-bound IRPs. Comparing the heme environmental structures surrounding HRMs, the flexible conformation and many amino acid residues sensitive to ROS of the IDD domain were suggested to promote specific oxidation by the generated hydrogen peroxide. Thus, heme binding to HRM near the IRE binding site sterically interferes with IRE binding, while HRM in the IDD domain facilitates specific heme-mediated oxidation of the protein moiety and the protein degradation via the ubiquitin-proteasome system, resulting in the inhibition of IRE binding.

Abstract P4-07-11: Investigating the activity of ESR1 allosteric compounds in ER+ breast cancer

Abstract Breast cancer (BCa) is the most frequently diagnosed cancer in women worldwide. Approximately 80% of breast cancer is estrogen receptor positive (ER+) with 74% demonstrating high expression of estrogen receptor alpha (). Thus, current endocrine therapies such as selective estrogen receptor degraders (i.e.- Fulvestrant) or selective estrogen receptor modulators (i.e-Tamoxifen) are involved modulation of ER signaling. However, most patients develop resistance to these drugs, and disease progression is common resulting in metastatic disease. Therefore, targeting alternative sites on the surface of has been proposed as an effective therapy to directly block its activity. In the present study, we investigated putative AF2 compounds that can bind to AF2 allosteric sites and inhibit complex formation and transcription of targetgenes. In the present study, we characterized a series of allosteric compounds using breast cancer cellular assays. MCF7 parental cells were treated with a series of putative allosteric compounds in a seven day CTG cell proliferation assay, and the compound ERX-11 was found to exhibit anti-proliferative activity. Genomic RNA was isolated after overnight treatment with ERX-11 to investigate compounds treatment on the expression of key ER pathway transcription factors via real time PCR. The degradation effect on nuclear ER and cytoplasmic ER after treatment with ERX-11 were assessed via western blots. We successfully characterized a series of putative allosteric compounds with ERX-11 being the only compound that exhibited anti-proliferative activity in both MCF7 parental and LSZ resistant cells. ERX-11 inhibited the activation of ER transcription gene and degradation of nuclear ER. These findings show promising effects of ERX-11 to block the AF2 sites on the binding domain of ERα with potential therapeutics for ER+ breast cancer. Citation Format: Uwimana A, Tiong Yip CL, Das R, Cheng Q, Meredith E, Gaither AL. Investigating the activity of ESR1 allosteric compounds in ER+ breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-07-11.