The entropic force generated by intrinsically disordered segments tunes protein function.

Protein structures are dynamic and can explore a large conformational landscape1,2. Only some of these structural substates are important for protein function (such as ligand binding, catalysis and regulation)3-5. How evolution shapes the structural ensemble to optimize a specific function is poorly understood3,4. One of the constraints on the evolution of proteins is the stability of the folded 'native' state. Despite this, 44% of the human proteome contains intrinsically disordered peptide segments greater than 30 residues in length6, the majority of which have no known function7-9. Here we show that the entropic force produced by an intrinsically disordered carboxy terminus (ID-tail) shifts the conformational ensemble of human UDP-α-D-glucose-6-dehydrogenase (UGDH) towards a substate with a high affinity for an allosteric inhibitor. The function of the ID-tail does not depend on its sequence or chemical composition. Instead, the affinity enhancement can be accurately predicted based on the length of the intrinsically disordered segment, and is consistent with the entropic force generated by an unstructured peptide attached to the protein surface10-13. Our data show that the unfolded state of the ID-tail rectifies the dynamics and structure of UGDH to favour inhibitor binding. Because this entropic rectifier does not have any sequence or structural constraints, it is an easily acquired adaptation. This model implies that evolution selects for disordered segments to tune the energy landscape of proteins, which may explain the persistence of intrinsic disorder in the proteome.

Dihydroquinazolines enhance 20S proteasome activity and induce degradation of α-synuclein, an intrinsically disordered protein associated with neurodegeneration.

Aggregates or oligomeric forms of many intrinsically disordered proteins (IDPs), including α-synuclein, are hallmarks of neurodegenerative diseases, like Parkinson's and Alzheimer's disease, and key contributors to their pathogenesis. Due to their disordered nature and therefore lack of defined drug-binding pockets, IDPs are difficult targets for traditional small molecule drug design and are often referred to as "undruggable". The 20S proteasome is the main protease that targets IDPs for degradation and therefore small molecule 20S proteasome enhancement presents a novel therapeutic strategy by which these undruggable IDPs could be targeted. The concept of 20S activation is still relatively new, with few potent activators having been identified thus far. Herein, we synthesized and evaluated a library of dihydroquinazoline analogues and discovered several promising new 20S proteasome activators. Further testing of top hits revealed that they can enhance 20S mediated degradation of α-synuclein, the IDP associated with Parkinson's disease.

Targeting intrinsically disordered proteins involved in cancer.

Intrinsically disordered proteins (IDPs) do not have a well-defined structure under physiological conditions, but they have key roles in cell signaling and regulation, and they are frequently related to the development of diseases, such as cancer and other malignancies. This has converted IDPs in attractive therapeutic targets; however, targeting IDPs is challenging because of their dynamic nature. In the last years, different experimental and computational approaches, as well as the combination of both, have been explored to identify molecules to target either the hot-spots or the allosteric sites of IDPs. In this review, we summarize recent developments in successful targeting of IDPs, all of which are involved in different cancer types. The strategies used to develop and design (or in one particular example, to repurpose) small molecules targeting IDPs are, in a global sense, similar to those used in well-folded proteins: (1) screening of chemically diverse or target-oriented compound libraries; or (2) study of the interfaces involved in recognition of their natural partners, and design of molecular candidates capable of binding to such binding interface. We describe the outcomes of using these approaches in targeting IDPs involved in cancer, in the view to providing insight, to target IDPs in general. In a broad sense, the designed small molecules seem to target the most hydrophobic regions of the IDPs, hampering macromolecule (DNA or protein)-IDP interactions; furthermore, in most of the molecule-IDP complexes described so far, the protein remains disordered.

Intrinsically Disordered Proteins as Regulators of Transient Biological Processes and as Untapped Drug Targets.

Intrinsically disordered proteins (IDPs) are critical players in the dynamic control of diverse cellular processes, and provide potential new drug targets because their dysregulation is closely related to many diseases. This review focuses on several medicinal studies that have identified low-molecular-weight inhibitors of IDPs. In addition, clinically relevant liquid-liquid phase separations-which critically involve both intermolecular interactions between IDPs and their posttranslational modification-are analyzed to understand the potential of IDPs as new drug targets.

Sesamin extends lifespan through pathways related to dietary restriction in Caenorhabditis elegans.

PURPOSE: Sesamin, a polyphenolic compound found in sesame seeds, has been reported to exert a variety of beneficial health effects. We have previously reported that sesamin increases the lifespan of Caenorhabditis elegans. In this study, we investigated the molecular mechanisms underlying the longevity effect of sesamin in C. elegans. METHODS: Starting from three days of age, Caenorhabditis elegans animals were fed a standard diet alone or supplemented with sesamin. A C. elegans genome array was used to perform a comprehensive expression analysis. Genes that showed differential expression were validated using real-time PCR. Mutant or RNAi-treated animals were fed sesamin, and the lifespan was determined to identify the genes involved in the longevity effects of sesamin. RESULTS: The microarray analysis revealed that endoplasmic reticulum unfolded protein response-related genes, which have been reported to show decreased expression under conditions of SIR-2.1/Sirtuin 1 (SIRT1) overexpression, were downregulated in animals supplemented with sesamin. Sesamin failed to extend the lifespan of sir-2.1 knockdown animals and of sir-2.1 loss-of-function mutants. Sesamin was also ineffective in bec-1 RNAi-treated animals; bec-1 is a key regulator of autophagy, and is necessary for longevity induced by sir-2.1 overexpression. Furthermore, the heterozygotic mutation of daf-15, which encodes the target of rapamycin (TOR)-binding partner Raptor, abolished lifespan extension by sesamin. Moreover, sesamin did not prolong the lifespan of loss-of-function mutants of aak-2, which encodes the AMP-activated protein kinase (AMPK). CONCLUSIONS: Sesamin extends the lifespan of C. elegans through several dietary restriction-related signaling pathways, including processes requiring SIRT1, TOR, and AMPK.

Pathway of binding of the intrinsically disordered mitochondrial inhibitor protein to F1-ATPase.

The hydrolysis of ATP by the ATP synthase in mitochondria is inhibited by a protein called IF1. Bovine IF1 has 84 amino acids, and its N-terminal inhibitory region is intrinsically disordered. In a known structure of bovine F1-ATPase inhibited with residues 1-60 of IF1, the inhibitory region from residues 1-50 is mainly α-helical and buried deeply at the α(DP)ß(DP)-catalytic interface, where it forms extensive interactions with five of the nine subunits of F1-ATPase but mainly with the ß(DP)-subunit. As described here, on the basis of two structures of inhibited complexes formed in the presence of large molar excesses of residues 1-60 of IF1 and of a version of IF1 with the mutation K39A, it appears that the intrinsically disordered inhibitory region interacts first with the αEßE-catalytic interface, the most open of the three catalytic interfaces, where the available interactions with the enzyme allow it to form an α-helix from residues 31-49. Then, in response to the hydrolysis of an ATP molecule and the associated partial closure of the interface to the αTPßTP state, the extent of the folded α-helical region of IF1 increases to residues 23-50 as more interactions with the enzyme become possible. Finally, in response to the hydrolysis of a second ATP molecule and a concomitant 120° rotation of the γ-subunit, the interface closes further to the α(DP)ß(DP)-state, allowing more interactions to form between the enzyme and IF1. The structure of IF1 now extends to its maximally folded state found in the previously observed inhibited complex.

Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

Dynamic Autoinhibition of the HMGB1 Protein via Electrostatic Fuzzy Interactions of Intrinsically Disordered Regions.

Highly negatively charged segments containing only aspartate or glutamate residues ("D/E repeats") are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40-900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.

Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

Role and therapeutic potential of liquid-liquid phase separation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease selectively affecting motor neurons, leading to progressive paralysis. Although most cases are sporadic, ∼10% are familial. Similar proteins are found in aggregates in sporadic and familial ALS, and over the last decade, research has been focused on the underlying nature of this common pathology. Notably, TDP-43 inclusions are found in almost all ALS patients, while FUS inclusions have been reported in some familial ALS patients. Both TDP-43 and FUS possess 'low-complexity domains' (LCDs) and are considered as 'intrinsically disordered proteins', which form liquid droplets in vitro due to the weak interactions caused by the LCDs. Dysfunctional 'liquid-liquid phase separation' (LLPS) emerged as a new mechanism linking ALS-related proteins to pathogenesis. Here, we review the current state of knowledge on ALS-related gene products associated with a proteinopathy and discuss their status as LLPS proteins. In addition, we highlight the therapeutic potential of targeting LLPS for treating ALS.

Identification of synthetic inhibitors for the DNA binding of intrinsically disordered circadian clock transcription factors.

Essential components of the human circadian clock, BMAL1 and CLOCK, which are intrinsically disordered transcription factors, were expressed and subjected to a fluorescent in vitro binding assay using an E-box DNA fragment. Screening of a chemical library identified 5,8-quinoxalinedione (1), which was found to inhibit binding of the heterodimer BMAL1/CLOCK to E-box at low micromolar concentrations.

Recent Advances in Computational Protocols Addressing Intrinsically Disordered Proteins.

Intrinsically disordered proteins (IDP) are abundant in the human genome and have recently emerged as major therapeutic targets for various diseases. Unlike traditional proteins that adopt a definitive structure, IDPs in free solution are disordered and exist as an ensemble of conformations. This enables the IDPs to signal through multiple signaling pathways and serve as scaffolds for multi-protein complexes. The challenge in studying IDPs experimentally stems from their disordered nature. Nuclear magnetic resonance (NMR), circular dichroism, small angle X-ray scattering, and single molecule Förster resonance energy transfer (FRET) can give the local structural information and overall dimension of IDPs, but seldom provide a unified picture of the whole protein. To understand the conformational dynamics of IDPs and how their structural ensembles recognize multiple binding partners and small molecule inhibitors, knowledge-based and physics-based sampling techniques are utilized in-silico, guided by experimental structural data. However, efficient sampling of the IDP conformational ensemble requires traversing the numerous degrees of freedom in the IDP energy landscape, as well as force-fields that accurately model the protein and solvent interactions. In this review, we have provided an overview of the current state of computational methods for studying IDP structure and dynamics and discussed the major challenges faced in this field.

Rational discovery of antimetastatic agents targeting the intrinsically disordered region of MBD2.

Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.

Hitting on the move: Targeting intrinsically disordered protein states of the MDM2-p53 interaction.

Intrinsically disordered proteins are an emerging class of proteins without a folded structure and currently disorder-based drug targeting remains a challenge. p53 is the principal regulator of cell division and growth whereas MDM2 consists its main negative regulator. The MDM2-p53 recognition is a dynamic and multistage process that amongst other, employs the dissociation of a transient α-helical N-terminal ''lid'' segment of MDM2 from the proximity of the p53-complementary interface. Several small molecule inhibitors have been reported to inhibit the formation of the p53-MDM2 complex with the vast majority mimicking the p53 residues Phe19, Trp23 and Leu26. Recently, we have described the transit from the 3-point to 4-point pharmacophore model stabilizing this intrinsically disordered N-terminus by increasing the binding affinity by a factor of 3. Therefore, we performed a thorough SAR analysis, including chiral separation of key compound which was evaluated by FP and 2D NMR. Finally, p53-specific anti-cancer activity towards p53-wild-type cancer cells was observed for several representative compounds.

Trimethylamine N-oxide abolishes the chaperone activity of α-casein: an intrinsically disordered protein.

Osmolytes (small molecules that help in circumventing stresses) are known to promote protein folding and prevent aggregation in the case of globular proteins. However, the effect of such osmolytes on the structure and function of intrinsically disordered proteins (IDPs) has not been clearly understood. Here we have investigated the effect of methylamine osmolytes on α-casein (an IDP present in mammalian milk) and discovered that TMAO (Trimethylamine-N-oxide) but not other methylamines renders α-casein functionless. We observed that the loss of chaperone activity of α-casein in presence of TMAO was due to the induction of an unstable aggregation-prone intermediate. The results indicate that different osmolytes may have different structural and functional consequences on IDPs, and therefore might have clinical implications for a large number of human diseases (e.g., amyloidosis, cancer, diabetes, and neurodegeneration) where IDPs are involved.

Structure-based Inhibitor Design for the Intrinsically Disordered Protein c-Myc.

Intrinsically disordered proteins (IDPs) are associated with various diseases and have been proposed as promising drug targets. However, conventional structure-based approaches cannot be applied directly to IDPs, due to their lack of ordered structures. Here, we describe a novel computational approach to virtually screen for compounds that can simultaneously bind to different IDP conformations. The test system used c-Myc, an oncoprotein containing a disordered basic helix-loop-helix-leucine zipper (bHLH-LZ) domain that adopts a helical conformation upon binding to Myc-associated factor X (Max). For the virtual screen, we used three binding pockets in representative conformations of c-Myc370-409, which is part of the disordered bHLH-LZ domain. Seven compounds were found to directly bind c-Myc370-409 in vitro, and four inhibited the growth of the c-Myc-overexpressing cells by affecting cell cycle progression. Our approach of IDP conformation sampling, binding site identification, and virtual screening for compounds that can bind to multiple conformations provides a useful strategy for structure-based drug discovery targeting IDPs.

Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson's disease.

The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aß peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.