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Intrinsically disordered proteins as potential drug targets


Intrinsically disordered proteins (IDPs) are a class of proteins that do not adopt a stable secondary or tertiary structure under physiological conditions in vitro, but still have biological functions. Many IDPs are implicated in cancer, neurodegenerative diseases, and diabetes, which makes them attractive drug targets. Are there any successful examples of that? Are there any drugs which disrupt protein-protein interactions (as one of the protein being IDP)?


IDPs are indeed attractive drug targets and there are ongoing efforts to develop drug molecules that block interactions between a disordered and a structured protein. According to this relatively recent paper, however, these efforts have not brought a drug on the market, yet.

A few promising studies have shown drug-like molecules that inhibit protein-protein interactions based on intrinsic disorder of one of the partners and target:

  • oncogenic fusion protein EWS-FLI1 and RNA helicase A. A small molecule has been found that targets the disordered region of EWS-FLI1, blocks the interaction with the helicase and inhibits growth of Ewing's sarcoma.

  • p53 tumor supressor and its interactor Mdm2. Mdm2, by binding to an intrinsically disordered region of p53, targets p53 for ubiquitination and also causes it to be transported out of the nucleus. Promising small molecules have been found that associate with Mdm2 and thereby block its interaction with p53.

  • c-Myc oncoprotein and the interaction with its partner Max protein. This study demonstrates two small molecules that bind to c-Myc and stabilize its disordered conformation, which inhibits its interaction with the Max protein.

The challenge in targeting protein-protein interactions for therapies stems largely from the fact, that the protein-protein contact surfaces are much larger than those involved in protein-small-molecule interactions (1,500-3,000 Å2 and (300-1,000 Å2, respectively) [2]. They are often flat and have no defined binding pocket. Also, IDPs often don't bind natural small ligands, that could act as starting points in developing drugs.

You may find this paper helpful:

Metallo SJ, Intrinsically disordered proteins are potential drug targets, Curr Opin Chem Biol. 2010 14(4): 481-488.

BTW: for a comprehensive, manually curated list of disordered proteins and regions, please check the Database of Protein Disorder.


I have been working on this problem for quite some time now and believe me getting specific binding is a real issue with IDRs. Also, since these regions dont form core structure of the protein, the residues are less conserved (more prone to mutations). So in case of evolving drug-resistance contributing proteins this become a bottleneck.

I have done some work on IDPs as potential drug target: Unraveling the potential of intrinsically disordered proteins as drug targets: application to Mycobacterium tuberculosis.

But the concept has been taken well by even the conventional structural biologists.

As the paper suggests, my work is focused on Mycobacterium tuberculosis. Multiple Drug resistance (MDR) and extremely drug resistance (XDR) form of TB have added to the tuberculosis loads not only in the developing but also developed nations. The drug resistance has been associated with proteins (mostly those involved in transport and metabolism) which have helped the pathogen grow resistant to first line of drugs or more. I refer such proteins as "Drug resistance associated proteins".


Article information

Unraveling the potential of intrinsically disordered proteins as drug targets: application to Mycobacterium tuberculosis

M. Anurag and D. Dash, Mol. BioSyst., 2009, 5, 1752 DOI: 10.1039/B905518P

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Intrinsically disordered proteins as potential drug targets - Biology

Hao Ruan, a Chen Yu, a Xiaogang Niu, bc Weilin Zhang, a Hanzhong Liu, d Limin Chen, e Ruoyao Xiong, a Qi Sun, a Changwen Jin, bc Ying Liu* ad and Luhua Lai /> * ade

a BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
E-mail: [email protected]
Tel: +861062757486

b College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China

c Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China

d Center for Quantitative Biology, Academy of Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China
E-mail: [email protected]
Tel: +861062751490

e Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China

Abstract

Intrinsically disordered proteins or intrinsically disordered regions (IDPs) have gained much attention in recent years due to their vital roles in biology and prevalence in various human diseases. Although IDPs are perceived as attractive therapeutic targets, rational drug design targeting IDPs remains challenging because of their conformational heterogeneity. Here, we propose a hierarchical computational strategy for IDP drug virtual screening (IDPDVS) and applied it in the discovery of p53 transactivation domain I (TAD1) binding compounds. IDPDVS starts from conformation sampling of the IDP target, then it combines stepwise conformational clustering with druggability evaluation to identify potential ligand binding pockets, followed by multiple docking screening runs and selection of compounds that can bind multi-conformations. p53 is an important tumor suppressor and restoration of its function provides an opportunity to inhibit cancer cell growth. TAD1 locates at the N-terminus of p53 and plays key roles in regulating p53 function. No compounds that directly bind to TAD1 have been reported due to its highly disordered structure. We successfully used IDPDVS to identify two compounds that bind p53 TAD1 and restore wild-type p53 function in cancer cells. Our study demonstrates that IDPDVS is an efficient strategy for IDP drug discovery and p53 TAD1 can be directly targeted by small molecules.


Proteus: a random forest classifier to predict disorder-to-order transitioning binding regions in intrinsically disordered proteins

The focus of the computational structural biology community has taken a dramatic shift over the past one-and-a-half decades from the classical protein structure prediction problem to the possible understanding of intrinsically disordered proteins (IDP) or proteins containing regions of disorder (IDPR). The current interest lies in the unraveling of a disorder-to-order transitioning code embedded in the amino acid sequences of IDPs/IDPRs. Disordered proteins are characterized by an enormous amount of structural plasticity which makes them promiscuous in binding to different partners, multi-functional in cellular activity and atypical in folding energy landscapes resembling partially folded molten globules. Also, their involvement in several deadly human diseases (e.g. cancer, cardiovascular and neurodegenerative diseases) makes them attractive drug targets, and important for a biochemical understanding of the disease(s). The study of the structural ensemble of IDPs is rather difficult, in particular for transient interactions. When bound to a structured partner, an IDPR adapts an ordered conformation in the complex. The residues that undergo this disorder-to-order transition are called protean residues, generally found in short contiguous stretches and the first step in understanding the modus operandi of an IDP/IDPR would be to predict these residues. There are a few available methods which predict these protean segments from their amino acid sequences however, their performance reported in the literature leaves clear room for improvement. With this background, the current study presents 'Proteus', a random forest classifier that predicts the likelihood of a residue undergoing a disorder-to-order transition upon binding to a potential partner protein. The prediction is based on features that can be calculated using the amino acid sequence alone. Proteus compares favorably with existing methods predicting twice as many true positives as the second best method (55 vs. 27%) with a much higher precision on an independent data set. The current study also sheds some light on a possible 'disorder-to-order' transitioning consensus, untangled, yet embedded in the amino acid sequence of IDPs. Some guidelines have also been suggested for proceeding with a real-life structural modeling involving an IDPR using Proteus.

Keywords: Disorder-to-order transition Intrinsic disorder Protean Random forest Topography length.

Figures

Distribution of size of the…

Distribution of size of the ‘annotated’ protean segments. The distribution is obtained from…

Amino acid propensities in the…

Amino acid propensities in the predicted A disordered versus B ordered regions. The…

Amino acid propensities in the…

Amino acid propensities in the annotated A protean versus B non-protean segments. The…

Secondary structural probabilities in the…

Secondary structural probabilities in the predicted A disordered versus B ordered regions. H,…

Secondary structural probabilities in the…

Secondary structural probabilities in the originally classified A protean versus B non-protean segments.…

Indecisiveness in adapting a particular…

Indecisiveness in adapting a particular secondary structure for the originally classified protean versus…

Recall versus precision curves to…

Recall versus precision curves to analyze the cross-validated performance of Proteus. AUC denotes…

Relative feature importance. The top…

Relative feature importance. The top ten features contributing most to the prediction in…

Analysis of Proteus score for…

Analysis of Proteus score for the cross-validated predictions. a Proteus score versus PPV…

Comparison of Proteus with other…

Comparison of Proteus with other classifiers using the standard evaluation measures. All methods…


Early progress reported in designing drugs that target 'disordered' proteins

St. Jude Children's Research Hospital scientists have identified a small, drug-like molecule that inhibits the function of a "disordered" protein in research that may advance a novel approach to hearing restoration. The study appeared recently in the journal Scientific Reports.

The protein, p27, is among the estimated one-third of human proteins called intrinsically disordered proteins (IDPs) that do not spontaneously fold into specific 3-D shapes. p27 helps to regulate cell division reduced levels of the protein are associated with the spread of breast and other cancers. This study, however, was sparked by evidence of the possible benefits of inhibiting p27, particularly to aid regeneration of sensory hair cells of the inner ear to combat hearing loss.

The results also raise broader hopes regarding drug development targeting disordered proteins. Disordered proteins are implicated in a wide range of diseases, including diabetes and neurodegenerative disorders, but so far drug-development efforts have failed. Most drugs work by binding to proteins' stable 3-D shape, which disordered proteins lack.

The p27 protein works by binding to an enzyme and forming a complex that blocks cell division. This type of regulation is necessary to keep cells in check when not otherwise instructed to divide. In this study St. Jude researchers used NMR spectroscopy to identify 36 small molecules that bind to two different but partially overlapping regions of p27 where the protein binds to the enzyme. NMR spectroscopy uses magnetic properties of atoms to discover structural details of different molecules and especially how they interact with one another. Most drugs are small molecules. One of the small molecules in this study inhibited p27 function in biochemical assays, demonstrating in principle that small molecules can disrupt and possibly regulate function of disordered proteins.

"The thought had been that small molecules would not bind specifically to disordered proteins," said co-corresponding author Richard Kriwacki, Ph.D., a member of the Department of Structural Biology. "This study demonstrates that small molecules identified by screening a library of compounds not only bind to a disordered protein, but sequester and inhibit the protein's activity."

Scientists have begun work to engineer a compound that forms a stronger bond and encompasses the p27-enzyme binding site.

"The concept of p27 inhibition as a possible strategy for hair cell regeneration has been around for more than 15 years, but until now no one has been able to do it," said co-corresponding author Jian Zuo, Ph.D., a member of the St. Jude Department of Developmental Neurobiology, who studies hair cell regeneration. "I knew Richard was an authority on intrinsically disordered proteins like p27 so I approached him and he came up with the innovative, some would say crazy, idea of screening small molecules for inhibition of p27."

Hair cells in the inner ear convert sound vibrations into electrical signals that travel to the brain via the auditory nerve. In chickens, fish and amphibians, hair cells regenerate from the surrounding cells called supporting cells, but human hair cells lost to injury, disease or age do not. Such damage is a leading cause of hearing loss.

Laboratory experiments have shown that supporting cells from mice can be coaxed into becoming hair cells in part by blocking production of p27.

Using NMR spectroscopy, researchers screened a library of small molecules for evidence of p27 binding that may disrupt protein function. Investigators were specially looking for small molecules that bind p27 where the protein normally binds its enzyme partner and blocks cell division. First author Luigi Iconaru, Ph.D., a St. Jude postdoctoral fellow, led the effort. Co-author Anang Shelat, Ph.D., assistant member of the St. Jude Department of Chemical Biology and Therapeutics, developed the small-molecule library using molecules that were slightly larger and more complex than traditionally used for drug- development screening.

Surprisingly, researchers found two distinct groups of small molecules that bind distinct, but overlapping segments of p27. The small molecules provided insight into how disordered proteins bind, including the dynamic interaction between small molecules and short-lived binding sites created by different arrangements of the amino acids that make up p27.

"The next step is to link the small molecules and binding sites identified in this study together to form larger compounds that bind p27 at multiple sites with greater affinity and specificity," Zuo said. "While small-molecule compounds are still a long way from the clinic, these results are another small step on the long road to a drug for hearing loss that could be infused into the cochlea to generate new hair cells."


Abstract

Many gene sequences in eukaryotic genomes encode entire proteins or large segments of proteins that lack a well-structured three-dimensional fold. Disordered regions can be highly conserved between species in both composition and sequence and, contrary to the traditional view that protein function equates with a stable three-dimensional structure, disordered regions are often functional, in ways that we are only beginning to discover. Many disordered segments fold on binding to their biological targets (coupled folding and binding), whereas others constitute flexible linkers that have a role in the assembly of macromolecular arrays.


MATERIALS AND METHODS

Study design

This study was designed to develop a novel platform for the discovery of drug leads based on molecular docking and MD simulations of the DOT-associated IDPRs of target proteins and, as a proof of concept, to identify candidate drugs, suppressing metastatic potentials of cancer cells in vitro and in vivo, by targeting an IDPR of MBD2 that undergoes a DOT upon association with its binding partner p66α for the integration of the Mi-2/NuRD CRC. These objectives were addressed by (i) analyzing intrinsic disorder predispositions of drug-target proteins and evaluating potential disorder-based binding regions (45), (ii) doing molecular docking with druggable compounds from the ZINC compound library to the potential drug-target sites, (iii) selecting two lead compounds based on the docking scores and off-target probabilities and experimental validation of target binding, (iv) evaluating the mode and efficiency of the compound binding via MD simulations, (v) assessing the identified leads for biological effects suppressing metastatic potentials of cancer cells, and (vi) verifying antimetastatic efficacy in a murine xenograft tumor model.

In animal studies, mice were randomly assigned to treatment and control groups. Numbers of tested mice were specified in each figure. Outliers were removed only if mice died at an early stage of the treatment according to the Hanyang University Institutional Animal Care and Use Committee (IACUC) dimension guideline. The primary end points were tumor size and cancer metastasis to lung. Mice were euthanized when moribund or at the end of the prespecified treatment period. All procedures were performed in accordance with institutional protocols approved by the IACUC of the Hanyang University. Pathology analysis was performed in a blinded fashion.

Statistical analysis

Data were presented as means ± SE. The sample size for each experiment, n, was included in Results and the associated figure legend. Everywhere in the text, the difference between two subsets of data was considered statistically significant if the one-tailed Student’s t test gave a significance level P (P value) less than 0.05. Multiple comparisons, more than two means, were performed using a univariate analysis of variance (ANOVA), where a Scheffe posttest was performed in some cases or Kruskal-Wallis test. GraphPad Prism was used to generate MI50 curves for cell lines treated with ABA and APC in vitro. In addition, IC50 curves for FRET assay were also generated by GraphPad Prism. Statistical analyses were performed using IBM SPSS statistics 23.


Discussion

Cavities in the rapidly fluctuating ensemble

The above analyses on IDPs were conducted in a manner similar to those on ordered proteins. Since IDPs exist in highly dynamic conformations, some concerns may be raised, which should be appropriately addressed.

One point is the suitability to define a cavity in the rapidly fluctuating ensemble of conformations that an IDP samples. The energy landscapes of IDPs are relatively flat and the conformations interconvert very fast, for example, in a timescale of nanosecond, which is much faster than the typical binding time of a ligand. Therefore, is it meaningful to define a cavity in IDPs? Would a predicted cavity wither away far before a ligand succeeds to bind it? The answer to dismiss such concern roots in the statistical thermodynamics: equilibrium population is governed by such laws as Boltzmann distribution and does not depend on the kinetic process. If a single conformation of protein can bind a ligand, the identical conformation in an ensemble can do the same thing although the conformation is now accompanied by a weight determined by the ensemble. Different kinetic schemes are possible in affording the thermodynamics. For example, after the ligand bind a short-lived conformation with an appropriate cavity, it may lock the protein in such a conformation, or, force the protein to jump among conformations with a similar cavity, but not to those with improper cavities. No matter how the kinetics comes out to be, the thermodynamics does not alter.

Another one is the suitability of the dataset of IDPs used. Some structures (although not all) in Disprot-pdb came from complexes by removing the partners. So one might question whether they can reflect the properties of IDPs in the disordered free form which would be the target. Here, we note that structures in pE-DB are mostly in the disordered free form, and the resulting difference with respective to ordered proteins is in the same direction with Disprot-pdb. Therefore, although the obtained quantitative values can not be considered accurate, the qualitative conclusions are likely reliable.

Examples of drug design of IDPs

IDPs are abundant in cells, but drug design where IDPs are the target remains an untapped source. Here we briefly survey examples of IDP drug design in the literature (Table 5) and discuss their druggability when data are available.

There are a few examples that are widely discussed in reviews, 6, 7, 15, 47-59 namely, p53-MDM2, c-Myc-Max, and EWS-Fli1. 20, 40, 45 The tumor-suppressor protein p53 is at the center of a large signaling network involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. 60, 61 The p53 protein is regulated by binding with multiple targets such as MDM2 and Taz2. 62 Small molecules have been screened to inhibit p53-MDM2 interaction and reactivate the p53 pathway in cancer cells. 40-42 These small molecules function by binding to MDM2 in the p53-binding pocket, but do not interact directly with p53. Therefore, this example belongs to “drug design involving IDPs,” but not “drug design targeting IDPs.” EWS-Fli1 and c-Myc-Max, on the other hand, belong to the latter case. EWS-Fli1 is an oncogenic fusion protein, which is exclusively present in Ewing's sarcoma family tumors. 20 C-Myc is a transcription factor that becomes active by forming a dimer with its partner protein Max, and is expressed constitutively in most cancer cells. 21 Both c-Myc and EWS-Fli1 are IDPs. By systematic screenings, small molecule inhibitors were identified that bind to c-Myc and EWS-Fli1 directly and prevent their interaction with partners. 20, 21, 43-48 The conformation ensemble of c-Myc370–409 in the unbound state has been characterized by MD simulations, 18 which was included in our pE-DB dataset. Five conformations with druggable cavities were identified in our analysis for c-Myc (Fig. 8). Hammoudeh et al. have experimentally identified the binding sites of different inhibitors in c-Myc. 21 These actual binding sites correlate well with the druggable cavities predicted by CAVITY (Fig. 8).

Five conformations of c-Myc370–409 with druggable cavity. Experimentally suggested binding sites (residues 374–385, and 402–409) are shown in spheres with rainbow colors, while other residues are shown in cartoon. The bottoms of the druggable cavity are depicted in dense gray lines. Graphics is prepared using PyMOL.

AF9 is a mixed lineage leukemia (MLL) fusion protein that causes oncogenic transformation of hematopoietic cells. 36 AF9 interacts with AF4, the most common fusion protein in acute leukemias. Bioinformatics analysis has revealed that fusion proteins are usually significantly enriched in structural disorder. 64 In the current example, both AF9 and AF4 are IDPs. Based on mapping studies, an AF4-derived peptide has been developed to specifically interact with AF9 and disrupt the AF4-AF9 interaction in vitro and in vivo. 22 The peptide induces necrotic cell death in leukemia cells and enhances the cytotoxic effect of established chemotherapeutic agents, holding promise as a component in the composite therapy for MLL leukemia. 49, 50 Recently, nonpeptidic inhibitors of AF9 were also successfully developed by a high-throughput screening assay. 51 AF9 was included in our Disprot-pdb dataset (PDB ID 2LM0). The 10 conformations of AF9 in the PDB afforded 55 cavities 5 of them were predicted to be druggable (Table 1). Therefore, AF9 is highly druggable.

PTP1B (protein-tyrosine phosphatase 1B) is a nontransmembrane enzyme found on the endoplasmic reticulum. It is a negative regulator of insulin and leptin signaling. PTP1B has been long recognized as a therapeutic target for diabetes and obesity. 65 In addition, it is overexpressed in breast tumors together with HER2, and its overexpression alone drives mammary tumorigenesis. Therefore, PTP1B acts also as a therapeutic target for mammary tumorigenesis and malignancy. PTP1B contains an ordered catalytic domain and a long disordered C terminus. Recently, an aminosterol natural product, trodusquemine (MSI-1436) was found to inhibit the enzyme function of PTP1B by binding to its disordered C terminus. 52 Interestingly, MSI-1436 works via an allosteric effect, that is, it binds two sites that are distinct from the active enzyme site and stabilizes an inactive conformation of PTP1B. This is in accordance with the suggestion that allostery has direct implications for the role of structural disorder in proteins and is thus helpful for the development of drugs and therapies. 66

Some other progress was achieved in targeting aggregating IDPs. 23, 59 Although the majority of IDPs have an inherent advantage in preventing aggregation, 32, 56 some “abnormal” IDPs are commonly found among proteins involved in amyloid formation and conformational diseases. The suppression of pathological amyloid fibril formation is an active area of research, and some strategies have been explored. For example, molecular tweezers were found to effectively perturb the aggregation processes via specific binding to lysine. 53-55 The known atomic structures of segments of amyloid fibrils were also used as templates in designing non-natural amino-acid inhibitors of amyloid fibril formation, 23 and a virtual screening was conducted on a subset of αSyn conformations to identify a ligand that is protective in cellular models of αSyn-mediated vesicular dysfunction. 56, 68

IDPs as potent drug targets

To be a potent drug target, the protein should not only have the potential to interact with designed small ligands, but should also possess an essential biological function and be closely related to diseases. Based on the druggable cavity probability of the proteins as discussed above and their biological importance in the literature, we provide a list of IDPs in Table 6 that are suitable targets for rational drug design. A few systems are discussed briefly as follows.

Adapter molecule crk (PDB ID 2EYY/2EYZ) is also known as proto-oncogene c-Crk or p38. It has several SH2 and SH3 domains and acts as an adaptor to link tyrosine kinases and small G proteins. It regulates transcription and cytoskeletal reorganization during cell growth, motility, proliferation, and apoptosis. 76 Increased expression of crk has been identified to be responsible for the malignant features of several human cancers including breast, ovarian, lung, brain, and stomach. Therefore, the inhibition of crk is an effective therapeutic means for the treatment of these malignancies. 76 For example, microRNAs have been used to decrease the translation of crk and effectively inhibit the invasion in non-small cell lung carcinoma cell lines. 81

p15 PAF (pE-DB ID 6AAA) is a proliferating cell nuclear antigen (PCNA) associated factor. 82 It is localized primarily in the nucleus and shares the conserved PCNA binding motif with several other PCNA binding proteins including CDK inhibitor p21. It also binds the transactivation region of p53 and strongly inhibits its transcriptional activity. The expression of p15 PAF in several types of tumor tissues was notably increased, especially in esophageal tumors. The structural characterization of human p15 PAF showed that it is an IDP with nonrandom structural preferences at sites of interaction with other proteins, 79 suggesting p15 PAF to be potential drug target.

p27 Kip1 (pE-DB ID 2AAA) is a human homologue of Sic1, both being pivotal CDK inhibitors and tight modulators of CDK-dependent phenotypes. p27 Kip1 mainly stops or slows down the cell division cycle, and thus plays an essential role in key cellular processes such as proliferation, differentiation and apoptosis. 83 If the expression of p27 Kip1 is reduced, the progression from G1 to S-phase becomes out of control, which facilitates the formation and growth of tumors. Therefore, p27 Kip1 is a tumor suppressor protein, and drugs able to protect/enhance the role of p27 Kip1 may be an effective means for anticancer strategies. 83 In this aspect, design of allosteric effectors (allosteric drugs) would be very useful, which has gaining a lot of momentum in traditional drug discovery. 84, 85 On the other hand, the downregulation of p27 Kip1 aids maintenance of stem cell pluripotency and tissue regeneration. 86 For example, p27 Kip1 inhibition therapy has been proposed for hearing restoration in mammals. Recently, a high-throughput screening strategy has been applied to successfully identify novel p27 Kip1 transcriptional inhibitors. 86

Optimism versus obstacles for drug design on IDPs

The results obtained in this study suggest that the druggability of IDPs may be comparable with that of ordered proteins. The average probability for cavities to be predicted druggable is 9% in IDPs, almost double the value found for ordered proteins (5%). Taking into consideration the high content of IDPs in various proteomes and their essential role in human diseases, we are optimistic on the design of drugs that target IDPs. Despite being in its infancy, the drug design against IDPs is in a continuous progress and essential advance has been achieved in a few cases. It is expected that the study will be extended and have a great future.

On the other hand, there are some obstacles for drug design targeting IDPs. The major obstacle is the lack of well-developed strategies. Traditional rational drug design against ordered proteins relies on the knowledge of the three-dimensional protein structure. However, IDPs usually exist in highly dynamic conformational ensembles, and accurate ensembles are difficult to determine via either experimental or theoretical means, which exclude traditional approaches in most cases. As proof, most cases of drug designs for IDPs were carried out by experimental screening, but not via rational design. An additional obstacle is the specificity/promiscuity. 32, 36, 87, 88 For IDPs with a determined conformational ensemble, a straightforward strategy of rational design is to extract metastable structures and then conduct traditional approaches, but the promiscuity would be serious because ligands bind to IDPs in a way of “ligand clouds around protein clouds.” 18 Therefore, the development of novel strategies is needed for better rational drug design on IDPs.


This work was supported by the Odysseus grant G.0029.12 (FWO, Research Foundation Flanders) to PT and by a VIB international postdoctoral ([email protected]) Marie-Curie COFUND fellowship for MG. WV acknowledges the Brussels Institute for Research and Innovation (Innoviris) grant BB2B 2010-1-12.

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Keywords: intrinsically disordered proteins, IDP ensembles, IDP function, disorder prediction, protein ensemble database

Citation: Varadi M, Vranken W, Guharoy M and Tompa P (2015) Computational approaches for inferring the functions of intrinsically disordered proteins. Front. Mol. Biosci. 2:45. doi: 10.3389/fmolb.2015.00045

Received: 29 May 2015 Accepted: 21 July 2015
Published: 05 August 2015.

Piero Andrea Temussi, Università di Napoli Federico II, Italy

Alfonso De Simone, Imperial College London, UK
Henriette Molinari, Istituto di Chimica delle Macromolecole ISMAC CNR, Italy
Tobias Madl, Medical University Graz, Austria

Copyright © 2015 Varadi, Vranken, Guharoy and Tompa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


Spectroscopic Screen

Kriwacki used nuclear magnetic resonance (NMR) spectroscopy to hunt for inhibitors of the cell cycle regulator p27 Kip1 . He and his colleagues first got interested in the protein because it’s highly expressed in inner-ear hair cells, preventing them from regenerating in people who’ve lost hearing due to loud noises or chemotherapy treatment. The protein is also involved in diabetes, obesity, and breast cancer.

Kriwacki’s team performed a fragment screen, looking for drug-like moieties that might bind to p27 Kip1 . (See “Piece By Piece,” The Scientist, June 2013.) But a standard 1,100-fragment library yielded only two hits, and Kriwacki suspected the usual-size fragments were simply too small to grab onto the flapping IDP. The key to success, he says, was creation of a specialized library, with fragments a bit larger. After screening a further 1,222 compounds from that library for interactions with p27 Kip1 , the researchers identified seven more hits. By computationally modeling those molecules and their interactions with the target p27 Kip1 , they found characteristics that allowed them to identify other possible interactors, which they confirmed with NMR. That brought the total number of hits to 36.

The researchers tested one of their hits using in vitro functional assays, and showed it was able to partially disengage p27 Kip1 from its cellular target, Cdk2/cyclin A—a displacement that activated the kinase. Theoretically, in a cell, this activation would lead to cell cycle progression (Sci Rep, 5:15686, 2015).

“The affinity is super-low it’s really just a proof-of-principle experiment,” says Kriwacki. By synthesizing larger second- and third-generation compounds, he says, the team is already seeing higher affinity of the small molecules for the IDP. Kriwacki says these compounds bind p27 Kip1 by a novel mechanism he expects to publish soon.

Pros
• One-dimensional NMR is sensitive enough to identify weak binding, says Kriwacki.
• Two-dimensional NMR can identify the binding sites for the hit molecules.

Con
• The method is expensive and time-consuming Kriwacki estimates the team used a month or more of continuous NMR time.


Figure 1

Figure 1. Growing interest of researchers in intrinsically disordered proteins. Number of publications (red bars) and corresponding citations (blue bars) related to IDPs by year, from 1991 to 2014. Publications and citations were retrieved from a search of WEB of Science (http://apps.webofknowledge.com) using IDP-related terms: “(intrinsically OR natively OR inherently) AND (disordered OR unfolded OR unstructured OR flexible) AND (protein OR proteins)”. Inset represents accumulative citations in each year.

The rapidly growing interest in IDPs can be attributed to several factors. The first of them is the role these proteins play in changing the understanding of the molecular mechanisms of protein action and in reshaping the protein structure–function relationship. The discovery of biologically active but extremely flexible proteins questioned the assumption that unique 3D structure is a prerequisite for protein function. Although IDPs lack stable structures at functional conditions, they are known to carry out a number of crucial biological functions that are complementary to the functional repertoire of structured (ordered) proteins. In any given organism, IDPs constitute a functionally broad and densely populated subset of its proteome. The overall biological importance of IDPs/IDPRs, and their crucial roles in many biological processes, are further supported by the evolutionary persistence of these proteins and regions. IDPs are common across the three domains of life, being especially abundant in the eukaryotic proteomes. Signaling motifs and sites of posttranslational modifications are commonly located within IDPRs, and disorder-based signaling and functioning are modulated by alternative splicing.

Second, IDPs are very attractive (and still poorly understood) subjects for theoretical and experimental characterization. For example, functional disorder-to-order transitions are very common in IDPs. Often, these transitions are coupled with the possibility that a single protein/region adopts different structures in complexes with different partners. Curiously, some ordered proteins require partial local unfolding and undergo order-to-disorder transitions to become functional, suggesting the existence of dormant functional disorder. The need for special means for the analysis of structural properties of IDPs/IDPRs, their conformational behavior, their intrinsically flexible states, the mechanisms of their interactions with various binding partners, and their highly diversified functional roles in biological systems all create a foundation for the explosion in the development of novel experimental and theoretical tools and approaches for the analysis of IDPs.

Biomedical aspects related to IDPs/IDPRs are also of great importance. In fact, intrinsic disorder is highly abundant among many proteins associated with various human diseases. Furthermore, IDPs are attractive drug targets and several small molecules have been shown to act by blocking protein–protein interactions that involve intrinsically disordered region of one of the partners.

Although IDPs are major players in cell signaling, regulation, and recognition, and although they are frequently involved in the pathogenesis of numerous human diseases, the phenomenon of protein intrinsic disorder was not mentioned in the major Biochemistry textbooks until quite recently. The significant achievements in this area of biochemistry, molecular biology, structural biology, and biophysics have been presented in several reviews spread among dozens of scientific journals and books. The aim of this issue on Intrinsically Disordered Proteins is to introduce several important aspects of protein intrinsic disorder. Most of the reviews are written by teams comprising several authorities in the corresponding field and young scientists. This approach provides the multiangular consideration of a given subject and defines the comprehensive, authoritative, and critical nature of these reviews.

First, Johnny Habchi, Peter Tompa, Sonia Longhi, and Vladimir Uversky have contributed a review that serves as a formal introduction to this thematic issue of Chemical Reviews on intrinsically disordered proteins. The authors start with a brief historical overview by showing the role of bioinformatics in establishing the IDP field. Then they provide a generalized description of the major computational and experimental tools which are used in the field for IDP analysis. They also discuss the variability of functional roles of IDPs, provide a brief analysis of the modes of IDP interactions with various binding partners, introduce the peculiarities of IDP evolution, discuss the abundance of IDPs in various proteomes, emphasize the roles of IDPs in the pathogenesis of various diseases, and stress the importance of IDPs as potential drug targets.

The idea of a complex nature of “simple” disordered proteins is developed in the review by Robin van der Lee, Marija Buljan, Benjamin Lang, Robert Weatheritt, Gary W. Daughdrill, A. Keith Dunker, Monika Fuxreiter, Julian Gough, Joerg Gsponer, David Jones, Philip Kim, Richard Kriwacki, Christopher Oldfield, Rohit Pappu, Peter Tompa, Vladimir Uversky, Peter Wright, and M. Madan Babu on classification of IDPs and IDPRs. These authors emphasize that structurally uncharacterized and currently nonannotated proteins and protein segments, which are commonly predicted to be disordered, represent a large source of functional novelty relevant for discovering new biology. The authors provide an important overview of the various classifications of IDPs and IDPRs that have been put forward in the literature and discuss diverse classification approaches based on function, functional elements, structure, sequence, protein interactions, evolution, regulation, and biophysical properties. They also suggest that combinations of multiple existing classification schemes provide a means to achieve high quality function prediction for IDPs and IDPRs, and potentially lead to the improved functional coverage and deeper understanding of protein function.

The task of a precise structural description of IDPs is extremely difficult because of the insufficient independent experimental measurements compared to the number of degrees of conformational freedom. Among numerous techniques suitable for structural characterization of IDPs and IDPRs in solution, nuclear magnetic resonance (NMR) spectroscopy is considered as one of the most powerful experimental approaches for gaining structural information about these highly flexible entities at atomic resolution under conditions that are close to physiological. In their NMR-centric review, Malene Jensen, Markus Zweckstetter, Jie-rong Huang, and Martin Blackledge report on the recent progress in the interpretation of experimental NMR data to gain crucial information for accurate delineation of the conformational space sampled by IDPs/IDPRs in their free and bound forms.

Peculiarities of protein structure and function are critically dependent on the environment. Even the most ordered proteins become unfolded under strong denaturing conditions. Many other environmental factors play crucial roles in controlling and regulating protein structure. A general property of every living organism is the complexity of its intracellular environment. In fact, proteins have evolved to function within cells, where the concentration of macromolecules, including proteins, nucleic acids, and carbohydrates within a cell can be as high as 400 g/L,(22) creating a crowded medium, with considerably restricted amounts of free water.(22-27) Obviously, “physiological conditions” commonly used in the majority of in vitro experiments, which are typically done at relatively ideal thermodynamic conditions of low protein and moderate salt concentrations, are a very poor model of the crowded cellular environment. Some crucial steps in making experimental environments more realistic include structural and functional analysis in the presence of model crowding agents, such as poly(ethylene glycol), dextran, Ficoll, inert proteins, etc.,(28, 29) or studying proteins directly inside the cell (e.g., by the in-cell NMR experiments that enable high-resolution investigations of proteins of interest directly in cellular environments(30)). Francois-Xavier Theillet, Andres Binolfi, Tamara Frembgen-Kesner, Karan Hingorani, Mohona Sarkar, Ciara Kyne, Conggang Li, Peter Crowley, Lila Gierasch, Gary Pielak, Adrian Elcock, Anne Gershenson, and Philipp Selenko analyze major physicochemical properties of cells and describe how IDPs might be affected by these various cellular properties.

The next three reviews of this issue are dedicated to the careful analysis of some intricate aspects of disorder-based functionality. First, Peter Tompa extends the concept of allostery to IDPs. Originally, allostery was introduced as a regulatory mechanism, where the activity of a protein is modified or regulated by the binding of a ligand to a site distant/different from the active site, thereby allowing such a protein to serve as an allosteric switch reacting to a specific signal. Since IDPs/IDPRs are often characterized by a combination of multiple regulatory sites, they can integrate and interpret multiple incoming signals. The author also suggests that structural disorder contributes to multisteric regulation by modular signaling proteins which are built as a combination of domains, motifs, and linkers, and can display complex regulatory behavior. Such multistericity explains the long-range flow of regulatory information resulting from the remodeling of the conformational ensemble of complex proteins and perfectly fits into the signaling networks of higher eukaryotes.

Next, Kim Van Roey, Bora Uyar, Robert Weatheritt, Holger Dinkel, Markus Seiler, Aidan Budd, Toby Gibson, and Norman Davey summarize the current state of the art in the field of short linear motifs, and they show that these ubiquitous and functionally diverse protein interaction modules are crucial functional elements of IDPs/IDPRs able to direct cell regulation.

In the subsequent review, Ursula Jakob, Richard Kriwacki, and Vladimir Uversky introduce conditionally and transiently disordered proteins, which undergo environment- or modification-induced order-to-disorder transitions crucial for their function, and which are reversed back to their ordered, nonfunctional state as soon as the environment is restored or the modification is removed. In other words, such proteins possess cryptic or dormant functional disorder which needs to be awoken in order to make these proteins active. A wide spectrum of factors grouped into two major classes, passive (i.e., environmental factors that are not dependent on any specific interaction between the protein and its partner) and active (i.e., factors that involve some specific interaction of a protein with its environment), can induce functional order-to-disorder transitions and activate corresponding proteins. Passive factors correspond to a modification of some global features of the protein environment, such as changes in pH, temperature, the redox potential, mechanical force, or light exposure, whereas active factors include interactions of a protein with membranes, ligands, other proteins, nucleic acids, or various post-translational modifications or release of autoinhibition.

The various roles of intrinsic disorder in assembly and function of proteinaceous machines are considered in the review by Monika Fuxreiter, Ágnes Tóth-Petróczy, Daniel Kraut, Andreas Matouschek, Roderick Lim, Bin Xue, Lukasz Kurgan, and Vladimir Uversky. The authors start with the consideration of intrinsic disorder as a crucial factor for the assembly of protein complexes and show that ordered complexes can be formed from the disordered monomers, that the presence of intrinsic disorder in monomers provides a means for stepwise and directional assembly, that such disorder-controlled stepwise assembly can be dependent on binding to some hidden sites, and that binding-induced (partial) folding of an IDP can generate a new conformation with a novel binding site, thereby providing a means for binding chain reactions. These general concepts are then illustrated by some specific examples of pliable proteinaceous machines, such as mediator, protein unfolding machines, nucleopore, ribonucleoprotein complexes, scaffold proteins, cytoskeleton, and extracellular matrix.

The review by Vladimir Uversky, Vrushank Davé, Lilia Iakoucheva, Prerna Malaney, Steven Metallo, Ravi Pathak, and Andreas Joerger considers different aspects of pathological unfoldomics and discusses the roles of intrinsically disordered proteins in the pathogenesis of human diseases. The authors emphasize that, since distortion of any of the mechanisms controlling IDP/IDPR functionality can be detrimental, IDPs/IDPRs are commonly found in various human diseases, ranging from cancer to cardiovascular disease, to neurodegenerative diseases, to diabetes. The involvement of IDPs in pathology is commonly associated with some abnormalities in their regulation, where chromosome translocation, aberrant splicing, and alternative splicing, altered expression, abnormal posttranslational modifications, and pathological mutations all might play a role. Two important cancer-related proteins, p53 and PTEN, are then used as illustrative examples of pathogenic IDPs. The last part of this review is dedicated to the consideration of IDPs as potential drug targets and to the introduction of currently available approaches for finding small molecules affecting the functions of IDPs/IDPRs.

The “IDPs in pathology” topic is continued by Bin Xue, David Blocquel, Johnny Habchi, Alexey Uversky, Lukasz Kurgan, Vladimir Uversky, and Sonia Longhi, who summarize the current knowledge on the abundance and roles of IDPs in viral proteomes. These authors discuss the unique origin and properties of viruses, provide a brief description of the classification of viral proteins, show which roles intrinsic disorder plays in structural proteins, focus on the multitude of functional roles of intrinsic disorder in nonstructural, regulatory, and accessory proteins, and discuss the role of intrinsic disorder in resolving potential structural chaos evoked by the use of common use alternative splicing and overlapping reading frames during the biosynthesis of viral proteins.

The review by Macarena Marı́n and Thomas Ott is dedicated to the abundance and roles of intrinsic disorder in plant proteins and phytopathogenic bacterial effectors. Among various aspects covered by this review are a general consideration of intrinsic disorder in plant proteins IDPs involved in abiotic stress response and plant signaling roles of disorder in chloroplast proteins involvement of IDPs/IDPRs in plant immunity and the roles of intrinsic disorder in effector proteins produced by plant pathogens, such as bacteria, fungi, oomycetes, and nematodes, to suppress or circumvent plant immune system.

Finally, the review by Remy Loris and Abel Garcia-Pino discusses intricate dynamics-based regulatory mechanisms in toxin-antitoxin (TA) modules, which are small operons that encode two genes, a toxic protein, and antitoxin protecting cells from this toxin. It is stated that the TA modules are ubiquitous in the genomes of prokaryotes and archeae. Depending on the molecular mechanisms of their action, TA modules can be grouped into three major types. The review covers the modular organization and origin of these modules and shows that intrinsic disorder is common in TA antitoxins, where it plays various functional roles. In fact, TA modules use IDPRs for regulation at the level of protein activity and transcription and exemplify the variety of functionalities that can arise from simple folding-upon-binding interactions.

In summary, this thematic issue provides a collection of focused articles in the field of intrinsically disordered proteins. These articles are not simply reviews, but they contain new interpretations and opinions of authors. Obviously, not all important aspects are covered, and this collection of reviews is meant to serve as a starting point for future discussions of this intriguing phenomenon. I hope that the conclusions and opinions collected in this thematic issue will serve as promoters of future research. Finally, I would like to acknowledge all the authors who contributed their time and put a lot of effort into bringing this project to fruition. I am also thankful to Prof. Robert Kuchta for the invitation to serve as the Guest Editor for this thematic issue. I am particularly indebted to Saundra Richter for her invaluable help at different stages of this project.

Views expressed in this editorial are those of the author and not necessarily the views of the ACS.