Development Of Protein-protein Interaction Inhibitors Research Articles

Maleimide constrained BAD BH3 domain peptides as BCL-xL Inhibitors: A versatile approach to rapidly identify sites compatible with peptide constraining

Development of protein–protein interaction (PPI) inhibitors remains a major challenge. A significant number of PPIs are mediated by helical recognition epitopes; although peptides derived from such epitopes are attractive templates for inhibitor design, they may not readily adopt a bioactive conformation, are susceptible to proteolysis and rarely elicit optimal cell uptake properties. Constraining peptides has therefore emerged as a useful method to mitigate against these liabilities in the development of PPI inhibitors. Building on our recently reported method for constraining peptides by reaction of dibromomaleimide derivatives with two cysteines positioned in an i and i + 4 relationship, in this study, we showcase the power of the method for rapid identification of ideal constraining positions using a maleimide-staple scan based on a 19-mer sequence derived from the BAD BH3 domain. We found that the maleimide constraint had little or a detrimental impact on helicity and potency in most sequences, but successfully identified i, i + 4 positions where the maleimide constraint was tolerated. Analyses using modelling and molecular dynamics (MD) simulations revealed that the inactive constrained peptides likely lose interactions with the protein as a result of introducing the constraint.

Getting a Grip on the Undrugged: Targeting β-Catenin with Fragment-Based Methods.

Aberrant WNT pathway activation, leading to nuclear accumulation of β‐catenin, is a key oncogenic driver event. Mutations in the tumor suppressor gene APC lead to impaired proteasomal degradation of β‐catenin and subsequent nuclear translocation. Restoring cellular degradation of β‐catenin represents a potential therapeutic strategy. Here, we report the fragment‐based discovery of a small molecule binder to β‐catenin, including the structural elucidation of the binding mode by X‐ray crystallography. The difficulty in drugging β‐catenin was confirmed as the primary screening campaigns identified only few and very weak hits. Iterative virtual and NMR screening techniques were required to discover a compound with sufficient potency to be able to obtain an X‐ray co‐crystal structure. The binding site is located between armadillo repeats two and three, adjacent to the BCL9 and TCF4 binding sites. Genetic studies show that it is unlikely to be useful for the development of protein–protein interaction inhibitors but structural information and established assays provide a solid basis for a prospective optimization towards β‐catenin proteolysis targeting chimeras (PROTACs) as alternative modality.

Phosphorylation of OCT4 Serine 236 Inhibits Germ Cell Tumor Growth by Inducing Differentiation.

Simple SummaryOctamer-binding transcription factor 4 (OCT4) plays an important role in early embryonic development, but is rarely expressed in adults. However, in many cancer cells, this gene is re-expressed, making the cancer malignant. This present study revealed that inhibiting OCT4 transcriptional activity induces cancer cell differentiation and growth retardation. Specifically, when the phosphorylation of OCT4 serine 236 increases by interfering with the binding of protein phosphatase 1 (PP1) to OCT4, OCT4 loses its transcriptional activity and cancer cells differentiate. Therefore, this study presents the basis for the development of protein-protein interaction inhibitors that inhibit the binding of OCT4 and PP1 for cancer treatment. Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.

Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors.

CBP (CREB (cAMP responsive element binding protein) binding protein (CREBBP)) and P300 (adenovirus E1A-associated 300 kDa protein) are two closely related histone acetyltransferases (HATs) that play a key role in the regulation of gene transcription. Both proteins contain a bromodomain flanking the HAT catalytic domain that is important for the targeting of CBP/P300 to chromatin and which offeres an opportunity for the development of protein-protein interaction inhibitors. Here we present the development of CBP/P300 bromodomain inhibitors with 2,3,4,5-tetrahydro-1,4-benzoxazepine backbone, an N-acetyl-lysine mimetic scaffold that led to the recent development of the chemical probe I-CBP112. We present comprehensive SAR of this inhibitor class as well as demonstration of cellular on target activity of the most potent and selective inhibitor TPOP146, which showed 134 nM affinity for CBP with excellent selectivity over other bromodomains.

Abstract 1358: Structure-based drug design of 3-site binding, high affinity inhibitors of S100B in malignant melanoma

Abstract An especially predictive biomarker when used in combination with other diagnostic indicators, S100B is elevated in >90% of malignant melanoma (MM) patients and its protein level correlates directly with poor survival (<1 yr.) and relapse. For patients with low levels of S100B (5-10%), MM vaccines are uniquely effective. While this correlation is not fully understood, mechanistic studies show that S100B is capable of blocking p53 oligomerization, promoting p53 phosphorylation in its C-terminus, and contributing to p53 degradation in concert with hdm2. Correspondingly, lowering S100B levels via siRNA or via small molecule inhibitors restore p53 levels and its tumor suppressor activities including UV-activated apoptosis pathways found in normal melanocytes. Additionally, elevated S100B and overexpression of the receptor for advanced glycation end products (RAGE) confers a metastatic phenotype. Studies into how S100B affects tumor progression and metastasis are ongoing, novel S100B inhibitors (termed SBiXs) to ablate S100B-dependent effects in cancer are in development. Structure-activity relationship (SAR) studies are ongoing to improve the affinity and selectivity of SBiXs. These studies reveal that the binding surface of S100B can be divided into “hot spots” or “persistent sites” that contribute more energetically to the protein-protein interactions (PPI) than the surrounding protein surface. Described here are descriptions of SBiXs which bind these Sites (1, 2, and 3), details to the SAR studies involved in the chemical optimization of these ligands, and information into the tethering of these SBiXs into a single-molecule capable of binding all 3 sites simultaneously. Also provided is a discussion regarding how protein dynamics affect the on/off rates of SBiXs and the resulting apparent binding affinity. The results described here represent a Structure-based drug design approach to the development of PPI inhibitors nearing the threshold required for a useful therapeutic window. Citation Format: Michael C. Cavalier, Paul T. Wilder, Diane Luci, David J. Maloney, Lei Fang, Mohd. Imran Ansari, Alexander D. MacKerell, Andrew Coop, Ajit Jadhav, David J. Weber. Structure-based drug design of 3-site binding, high affinity inhibitors of S100B in malignant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1358.

Investigating the dynamic aspects of drug-protein recognition through a combination of MD and NMR analyses: implications for the development of protein-protein interaction inhibitors.

In this paper, we investigate the dynamic aspects of the molecular recognition between a small molecule ligand and a flat, exposed protein surface, representing a typical target in the development of protein-protein interaction inhibitors. Specifically, we analyze the complex between the protein Fibroblast Growth Factor 2 (FGF2) and a recently discovered small molecule inhibitor, labeled sm27 for which the binding site and the residues mainly involved in small molecule recognition have been previously characterized. We have approached this problem using microsecond MD simulations and NMR-based characterizations of the dynamics of the apo and holo states of the system. Using direct combination and cross-validation of the results of the two techniques, we select the set of conformational states that best recapitulate the principal dynamic and structural properties of the complex. We then use this information to generate a multi-structure representation of the sm27-FGF2 interaction. We propose this kind of representation and approach as a useful tool in particular for the characterization of systems where the mutual dynamic influence between the interacting partners is expected to play an important role. The results presented can also be used to generate new rules for the rational expansion of the chemical diversity space of FGF2 inhibitors.

Complement-related Innovations: From the immunology laboratories to biotechnology companies

Event Abstract Back to Event Complement-related Innovations: From the immunology laboratories to biotechnology companies Kheir Eddine Kerboua1*, Warda Meziane2, Bey Baba-Hamed3, Amina Boumediene1 and Mourad Aribi2 1 Unite of Immunology, Military University Hospital of Oran,Algeria, Algeria 2 Laboratory of Applied Molecular Biology and Immunology. University of Tlemcen, Algeria 3 Department of Biotechnology of Oran, Algeria, Algeria Increased knowledge about the involvement of complement network in physiology, pathology and homeostasis,the development of high qualified research teams in structural and analytical techniques, and the promising experiences of Eculizumab and Mirococept in human medicine have resulted in thousands of creative approaches on Complement-related Innovations (CRI) in various immunological applications. This paperexposes the biotechnological attractiveness of the over fifty complement proteins in the development of potentially life-saving diagnostic and therapeutic tools that has lead to more than 1,000 patents related to the complement system. We alsohighlight questions about the safety and potential risks of therapeutic complement inhibition. Among the CRI, the development of protein-protein interaction inhibitors has been gaining traction, and monoclonal antibodies or antibody fragments are the most growing class in this field. In fact, many starts up have developed Minibodies (Adienne Laboratory), Aptamers (Ophthotech Laboratory) and Speigelmers (Noxxon laboratory) as alternative macromolecular blocking entities. On the other hand, we discuss the natural concepts-based CRI where biotechnological companies exploit the powerful evasion strategies of microorganisms and parasites to develop more promising drug candidates. Finally, selected examples of complement therapeutics on the market, on clinical trials and in pre-clinical development are highlighted to illustrate the creativity in field. Keywords: complement-related innovations, biotechnologicalmacromolecular blockers, risks of therapeutic complement inhibition, creative approaches on Complement-related Innovations (CRI), complement system Conference: The First International Congress of Immunology and Molecular Immunopathology (CIMIP2014), Tlemcen, Algeria, 17 Oct - 20 Oct, 2014. Presentation Type: Poster Presentation Topic: Immunodeficiencies Citation: Kerboua K, Meziane W, Baba-Hamed B, Boumediene A and Aribi M (2014). Complement-related Innovations: From the immunology laboratories to biotechnology companies. Front. Immunol. Conference Abstract: The First International Congress of Immunology and Molecular Immunopathology (CIMIP2014). doi: 10.3389/conf.fimmu.2014.04.00026 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Oct 2014; Published Online: 01 Dec 2014. * Correspondence: Dr. Kheir Eddine Kerboua, Unite of Immunology, Military University Hospital of Oran,Algeria, Oran, Algeria, K.K.Eddine@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kheir Eddine Kerboua Warda Meziane Bey Baba-Hamed Amina Boumediene Mourad Aribi Google Kheir Eddine Kerboua Warda Meziane Bey Baba-Hamed Amina Boumediene Mourad Aribi Google Scholar Kheir Eddine Kerboua Warda Meziane Bey Baba-Hamed Amina Boumediene Mourad Aribi PubMed Kheir Eddine Kerboua Warda Meziane Bey Baba-Hamed Amina Boumediene Mourad Aribi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Fine-mapping the Contact Sites of the Escherichia coli Cell Division Proteins FtsB and FtsL on the FtsQ Protein*

Escherichia coli cell division is effected by a large assembly of proteins called the divisome, of which a subcomplex consisting of three bitopic inner membrane proteins, FtsQ, FtsB, and FtsL, is an essential part. These three proteins, hypothesized to link cytoplasmic to periplasmic events during cell division, contain large periplasmic domains that are of major importance for function and complex formation. The essential nature of this subcomplex, its low abundance, and its multiple interactions with key divisome components in the relatively accessible periplasm make it an attractive target for the development of protein-protein interaction inhibitors. Although the crystal structure of the periplasmic domain of FtsQ has been solved, the structure of the FtsQBL complex is unknown, with only very crude indications of the interactions in this complex. In this study, we used in vivo site-specific photo cross-linking to probe the surface of the FtsQ periplasmic domain for its interaction interfaces with FtsB and FtsL. An interaction hot spot for FtsB was identified around residue Ser-250 in the C-terminal region of FtsQ and a membrane-proximal interaction region for both proteins around residue Lys-59. Sequence alignment revealed a consensus motif overlapping with the C-terminal interaction hot spot, underlining the importance of this region in FtsQ. The identification of contact sites in the FtsQBL complex will guide future development of interaction inhibitors that block cell division.

Identification of High Affinity Polo-like Kinase 1 (Plk1) Polo-box Domain Binding Peptides Using Oxime-Based Diversification

In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.

Abstract 1351: Computational design and synthesis of non-peptidic inhibitors targeting the cyclin binding groove of CDK2/Cyclin A

Abstract Inhibition of CDK2/Cyclin A (CDK2/CA) at G1/S phase of cell cycle controls cell proliferation and induces selective apoptosis of cancer cells. Neutralization of E2F-1by CDK2/CA phosphorylation is required prior to S-phase exit or apoptosis is triggered. E2F-1 is deregulated in many types of cancer and inhibition of CDK2/CA leads to selective cell death in these tumors. In our present study we target the substrate recruitment site knows as cyclin binding groove (CBG) for inhibition of CDK2/CA. The CBG recognizes a consensus sequence present on cyclin interacting proteins and the endogenous tumor suppressors knows cyclin binding motif (CBM) which has been minimized to the pentapeptide, RRLIF. It has been shown that the drug likeliness of the peptide can be improved by ligation of small molecules to truncated sequences. This was achieved using a unique strategy for designing inhibitors of protein-protein interactions known as REPLACE, in which small molecule fragments were identified by in silico methods. These were then ligated onto a truncated peptide sequence in order to replace critical residues, including the N-terminal Arginine moiety important for cyclin binding. Small molecule fragments for in silico docking were selected based on molecule weight, drug like features and possession of a carboxylic acid group for the formation of the peptide link. Docking was carried out using LigandFit and the crystal structure of 1-phenyl-1H-[1,2,4]triazole N-terminally capped peptides (Ncaps) in the CBG provided insight into molecular design. The 3,5 dichloro1-phenyl-1H-[1,2,4]triazole derivative linked to Arg-Leu-Asn-pF-Phe was previously shown to be a potent inhibitor with micromolar activity. In our present study we have replaced the N-terminal Arginine using different heterocyclic isosteres capable of interactions similar to critical amino acids of the parent peptide and the triazole. In order to probe the structure-activity relationship, we synthesized pyrazole, furan, pyrrole and thiazole systems and explored various substitutions of the phenyl ring. A number of Ncaps were then ligated to the tetra peptide, RLIF using solid phase synthesis, purified by reverse phase HPLC and characterized by MS. In vitro binding and functional assays were performed in order to study the inhibitory effect of compounds on CDK2/Cyclin A prior to further evaluation in cell viability assays to determine anti-tumor effects. On the basis of the results, further high throughput docking of potential heterocyclic fragments was carried out to identify N-capping groups of varying chemical diversity for synthesis and in vitro testing. The results presented demonstrate the utility of the REPLACE method in the development of protein-protein interaction inhibitors and non-ATP competitive CDK2 inhibitors as anti-tumor therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2011-1351

Protein-Protein Interaction Inhibitors: Small Molecules from Screening Techniques

Protein-protein interactions play crucial roles in a number of biological processes, and, as such, their disruption is becoming an area of intense research. Despite the many challenges associated with the development of protein-protein interaction inhibitors, such as the large and relatively featureless interfacial areas involved, there has been considerable success in recent years. Importantly, through the existence of protein "hot spots", some of this success takes the form of small molecule inhibitors that have been identified from a variety of screening techniques.