Small Molecule and Peptide Recognition of Protein Transmembrane

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Small molecule and peptide recognition of protein transmembrane domains Xianfeng Zeng, Peiyao Wu, Chengbo Yao, Jiaqi Liang, Shu-Ting Zhang, and HANG Hubert YIN Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00909 • Publication Date (Web): 29 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Small molecule and peptide recognition of protein transmembrane domains Xianfeng Zeng,1 Peiyao Wu,1 Chengbo Yao,1 Jiaqi Liang,1 Shuting Zhang,1,3* Hang Yin1,2* 1

Center of Basic Molecular Science, Department of Chemistry, Tsinghua University, Beijing 100082 China. 2

Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado Boulder, Boulder, CO 80309, USA.

3

School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China.

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Abstract: Membrane proteins play vital roles in cell signaling, molecular transportation and cell adhesion. The interactions of transmembrane domains are much less well understood than those of their water-soluble counterparts, and they have been deemed “undruggable” despite their important biological functions such as protein anchoring, signal transduction, and ligand recognition. Nevertheless, continual developments in this area have revealed useful probes for investigating and regulating these membrane proteins. This review summarizes and evaluates the strategies available for discovering small molecules and peptides that recognize the protein transmembrane domains of membrane proteins, with a particular focus on rational design and library screening.


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1. Introduction Membrane proteins (MPs) constitute about 23% of all human proteins and around 50% of membrane mass, and are involved in a huge number of crucial biological functions.1 Cell adhesion molecules such as integrins and selectins participate in cell communication between adjacent cells or between cells and the extracellular matrix, thus allowing cell migration and localization, which are key steps in oncogenesis and immune responses. Cell surface receptors such as epithelial growth factor receptors (EGFR), toll-like receptors (TLRs), and NOD-like receptors are able to distinguish molecule types and trigger downstream signaling pathways. For instance, EGFR undergoes a transition from an inactive monomeric form to an active homodimer upon EGF binding, and subsequently initiates downstream gene activation and signaling pathways. TLRs and NOD-like receptors which belong to the pattern recognition receptor family and trigger immune responses via the identification of structurally conserved molecules from microbes or stressed cells are termed as pathogen-associated molecular patterns and damage-associated molecular patterns, respectively. Membrane transporter proteins including carriers and channels are crucial for cell growth, homeostasis and the conduction of nerve pulses due to their ability to facilitate materials across the cellular membrane. Large groups of membrane-bound enzymes are also involved in various vital metabolic reactions. Therefore, MPs are critical targets in drug discovery due to their indispensability in biological functions and their dysregulation leads to severe cellular malfunctions and diseases. Transmembrane domains (TMDs) are fundamental in anchoring the MPs to the cellular surface, and the subsequent oligomerization is important in signal transduction and ligand recognition.2 Due to their crucial cell signaling transduction abilities, MPs make up approximately 60% of drug targets. However, targeting these TMDs with exogenous agents still remains a daunting challenge due to several reasons.3 First, conventional probes (e.g., antibodies) are implicated with loss of function within phospholipid bilayers due to the highly hydrophobic environment. Second, an appropriate level of hydrophobicity is required for the probes to properly interact with the lipid bilayer. Other properties such as hydrogen bonds, electrostatic interactions, and polarpolar interactions are also essential for TMD target selectivity and high affinity. Lastly, the binding of TMDs


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rarely takes place in distinguished pockets such as enzymes, making them difficult for small molecules to seek out. Despite these major hurdles, recent advances have been made in the discovery of TMD probes. Herein, we introduce two general strategies for the discovery of TMD targeting agents, focusing specifically on rational design and library screening (Figure 1).

Figure 1. Overviews of major methods to identify agents recognizing MP TMDs.

2. Rational design of agents targeting TMDs 2.1 Rational design based on natural product scaffolds Nature offers inspiration for rational drug design with bioactive molecules. Novel probes with a higher affinities or extended functions are obtained by modifying the structure of natural TMD-recognizing agents (Figure 2). While this approach has been fruitful, the main issue needed to be considered is that modification of natural scaffolds may affect the interactions between the molecule and its MP target.


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Figure 2. Rational design approaches based on natural product scaffolds. One of the approaches for the discovery of new agents recognizing protein TMDs is to attach a new pharmacophore to natural small molecules (left). The high affinity between nature-existed molecules and their TMD targets would guide the new attached pharmacophore to the binding sites; Also, structural modifications on natural small molecules could improve the affinity between those natural product analogs and TMD target (right).

Natural products are first modified to obtain probes designed to specifically target various TMD conformations. The discovery of specific bile acid receptor agonists exemplifies this kind of modification. Bile acids including cholic acid and lithocholic acid (LCA) are amphipathic steroid acids. They are recognized by nuclear bile acid receptors, such as the Farnesoid X receptor (FXR) and G protein-coupled bile acid receptor (GPBAR1, also known as TGR5). Insufficient bile acid signaling leads to adverse metabolic disorders such as cholesterol maladies and type II diabetes.4 Various modifications of bile acids have been explored to obtain drugs that can selectively activate one of the two receptors to reduce clinical side effects. Francesco et al. explored interactions between bile acid derivatives and human TGR5, and identified 3-amino-LCA derivatives 1-3 (Figure 3), a series of TGR5-specific agonists with low EC50 values. Homology modeling and in silico simulations predicted that the Glu169 anion within the TMD of hTGR5 interacts with the 3α-hydroxyl group in the LCA scaffold via hydrogen bonding, whereas in FXR, the positively charged His444 within the TMD interacts with the oxygen atom in 3α-hydroxyl in the LCA scaffold. Consequently, an ammonium cation was introduced at the C3 position of the LCA by replacing a primary amino group with a hydroxyl group, which successfully reduced its affinity toward FXR.5


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Figure 3. The structure of selective TGR5 agonists 1-3. Compounds 1-3 were obtained by replacing hydroxyl group with amino group in scaffold of LCA.

Linking various pharmacophores to the natural substrates of transporters is another common strategy for drug development. For example, a hexose-conjugate, with a combination of hexose moiety and anticancer pharmacophore, is selective toward cancer cells that recognize glucose transporters (GLUTs).6 GLUTs are vital in facilitating the transportation of glucose and other carbohydrates across cell membranes, a process that is especially important in cancer cells. Cancer cells generally show a low metabolic efficiency under anaerobic conditions, and require an increased level of hexose uptake to enable their unregulated growth (a.k.a. Warburg effect, Figure 4A).7, 8 As a consequence, high levels of hexose uptake and GLUTs expression are indicative of several types of cancer.9-11 Patra et al. designed three glucose-platinum conjugates that show great cytotoxicity toward cancer cells. Based on the crystal structure of XylE, a GLUT1 homolog, they conjectured that the hydroxyl group on the C6 of glucose is not an active participant in the glucose-GLUT recognition, and modification of this hydroxyl group might be tolerated in transporter recognition. The results of docking designed conjugates (Glc-Pts 4-6 Figure 4C) into XylE show that these conjugates form hydrogen bonds with various residues in XylE TMD. Further biochemical experiments indicated that these conjugates selectively accumulated in cancer cells (ovarian cancer cells A2780 and prostate cancer cells DU145) while having little impact on noncancerous neuron cells in vitro (Table 1).12 Similarly, Wu et al. reported galactose conjugated cisplatinum (Glc-Pts 7, Figure 4C) with promising efficacy against lung cancer and colon cancer.13


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Figure 4. Warburg effect and strategies of developing small-molecule drugs exploiting Warburg effect. (A) Warburg effect: GLUTs are overexpressed in cancer cells to compensate for low metabolic efficiency, and enable unregulated cell growth; (B) Strategies exploiting Warburg effect: Glucose conjugates enable selective delivery of anticancer pharmacophores to the cancer cells (left); GLUTs inhibitors can directly block the glucose uptake in energy demanding cancer cells (right); (C) The structure of designed hexose conjugates and their aglycone moiety.


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Table 1. Effects of Glc-Pts 4 and its aglycone moiety on the platinum uptake and reduction of cell viability in ovarian cancer cell line A2780 and prostate cancer cell line DU145. Uptake (pmol Pt/106 cells)

Reduction in cell viability (%)

A2780

DU145

A2780

DU145

4

19.0±0.7

116.6±4.3

44.7±1.2

58±3.5

aglycone

9.4±0.5

69±0.09

16.6±1.7

35±3.2

Certain antibiotics and cytotoxins regulate activities of enzyme and transporter via specific interactions of membrane-bound TMD.14 The identification of antibiotic and TMD interactions, followed by investigation of the structure-activity relationship (SAR) among the modified antibiotics, can be a useful strategy for overcoming multidrug resistance in certain types of bacteria. Bacterial lipoprotein signal peptidase II (LspA) is a key enzyme in the post-translational lipidation of bacterial proteins, and plays an important role in controlling the physiology and pathogenicity of bacteria.15 Globomycin 8 (Figure 5A), a 19-membered cyclic depsipeptidic antibiotic isolated from Streptomyces halstedii,16 can inhibit Gram-negative bacteria growth by interacting with LspA.17 Further research reveals that the disruption of the normal post-translational lipidation by 8 leads to the accumulation of diacylglyceride-containing lipoprotein precursors in the cytoplasmic membrane,18 which is toxic to bacteria. Kiho et al. conducted the SAR study of 8 and showed that the length of the alkyl side chains and the hydroxyl group in L-Serine are both essential for inhibitory activity.19 The alkyl chain modification resulted in an analog 9 (Figure 5A) that was 10 times more potent than 8. In addition, compound 9 showed a wider antibacterial spectrum than 8 (Table 2). This study exemplifies the viability of modifying antibiotics to counteract drug resistance. Vogeley et al. solved the crystal structure of the LspA complex from Pseudomonas aeruginosa with 8 at 2.8 Å resolution (Figure 5B).20 Using molecular dynamics simulation, they proposed that 8 interacted with LspA via hydrophobic and hydrogen bonding interactions with certain Asp residues (Asp124 and Asp143) that are responsible for catalyzing the signal peptide hydrolysis. The authors also pointed out that modification of the alkyl chain could strengthen the interactions between 8 analogs and the apolar surface of the


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MH2 transmembrane helix, leading to a higher affinity with analog 9.20 These results pave the way for the design of antibacterial drugs targeting LspA.

Figure 5. The molecular structure of globomycin and its co-crystal structure of globomycin in complex with LspA. (A) The structure of globomycin (8) and its analog 9; (B) The crystal structure of LspA-globomycin complex. (PDB entry: 5DIR). Modified from Reference 20. Figure 5B is reprinted with permission.

Table 2. Antimicrobial activity of globomycin (8) and its analog 9 against E. coli and S. aureus (MRSA). Minimum inhibitory concentration (MIC, ug/mL) E. coli

S. aureus (MRSA)

8

12.5

>100

9

1.56

12.5

2.2 Truncated peptides Interactions between TMDs contribute to the overall process of MP oligomerization. ToxR assay is generally applied to investigate the interactions between TMDs (Figure 6A). ToxR, derived from Vibrio cholerae, is a single-pass transmembrane activator located on the inner membranes of bacteria. Dimeric ToxR activates the ctx promoter and subsequently triggers reporter gene expression. By combining ToxR with various TMDs, the associations of TMDs can be evaluated by the expression level of the reporter gene. Langosch et al. first proposed ToxR assay and demonstrated that the GXXXG motif of GpA TMD was critical in its dimerization.21, 22

Similarly, Russ et al. used an alternative reporter, chloramphenicol acetyltransferase (CAT), and developed

the TOXCAT assay.23 Other reporter gene systems, such as lacZ and firefly luciferase, have also been applied to ACS Paragon Plus Environment

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ToxR systems.24 Dominant-negative TOXCAT (DN-TOXCAT), a variation of TOXCAT technique, is used to measure the heterodimerization of TMDs. One TMD is fused with a functional ToxR while a second TMD is fused with a mutant ToxR. Because only the dimer formed by two functional ToxR initiates a downstream CAT synthesis, the subsequent CAT signal correlates with the strength of TMD interaction. Although TOXCAT was originally designed to study single pass TMD dimerization, Yin and co-workers showed that it is also useful in investigating interactions of multipass transmembrane helices.25

Figure 6. TOXCAT system and truncated peptides as probes recognizing MP TMDs. (A) TOXCAT system: MBP: maltose binding protein; TM1 and TM2: Two engineered transmembrane helices; TM1 and TM2 interact with each other and form a functional ToxR dimer. The ToxR dimer binds to ctx promoter and induces measurable synthesis of CAT. TOXCAT provides a general way to determine the heterodimerization of two transmembrane helices; (B) Truncated peptides: Once the interacting protein transmembrane helices are determined, the truncated TMDs of one oligo partner can be exploited to modulate native oligomerization.


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Naturally existing interactions among TMD helices within MP oligomers can be exploited to develop truncated TMD peptides and to regulate the activities of the original MPs (Figure 6B). This approach, known as truncated peptides, allows discovery of peptides regulating MP oligomerization process, which has resulted in several useful probes for TLRs, insulin receptors (IRs), and Her (ErbB) family proteins. Compared to small-molecular probes, truncated peptides are selective and efficacious with less off-target effects. TLRs play critical roles in the innate immune system by specifically recognizing a variety of pathogen- and damage-associated molecular patterns.26 Among them, the heterodimerization between TLR2 and TLR1 or TLR6 is crucial for host immunity against lipopeptides anchored on the bacterial surface.27, 28 Using the ToxR assay, Godfroy et al. demonstrated the dimerization of various TLRs and their subsequent signaling transduction.29 Based on these studies, Fink et al. took advantage of the truncated TMDs of TLR2 and TLR6 to inhibit their dimerization and obstruct the subsequent signaling pathway.30 Due to the resemblance between TLR1 TMD and TLR6 TMD, both of these truncated TMD peptides are able to antagonize the secretion of proinflammatory cytokines such as TNF-α and interleukin 6 (IL-6) in macrophages after treatment of lipoteichoic acid (LTA, TLR 2/6 agonist) and Pam3CSK4 (TLR1/2 agonist), but not lipopolysaccharide (LPS, TLR4 agonist). Comprehensive biochemical and biophysical experiments have verified the interactions between these truncated peptides and their reciprocal TLRs. In vivo studies showed that truncated peptides derived from TLR1 and TLR6 were able to rescue mice with TLR2-related fatal inflammation, but not TLR4.29 Moreover, Shmuel-Galia et al. utilized this TLR2 truncated peptide to further investigate the role of TLR2 heterodimerization in the activation of pathogenic pro-inflammatory Ly6Chi monocytes and demonstrated the therapeutic potential of truncated TMD peptides in ameliorating colitis.31 Shoelson and co-workers applied this strategy of using TM domain sequence peptide to identify agonist of IR.32 IR is a member of receptor tyrosine kinase (RTK) family, and plays an essential role in cell metabolism and growth.33-35 Shoelson et al demonstrated that a 24-residue IR TMD containing oligopeptide can specifically activate IR signaling. Based on biochemical analyses, the dissociation of two inactive IR TMD dimers potentially leads to closer proximity of two β-subunits and initiates the IR activation.36, 37 Upon the binding of truncated IR-TMD peptide, the dimerization of two inactive IR TMDs collapses, resulting in the juxtaposition of the two β-subunits and IR auto-phosphorylation. ACS Paragon Plus Environment

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Due to their great importance in human cancer pathogenesis, the TMDs of the ErbB family proteins have been thoroughly studied.38-40 Truncated peptidic probes have also been involved in the study of the Her (ErbB) family of RTKs including ErbB1 (Her1/ EGFR), ErbB2 (Her2/ Neu), ErbB3, and ErbB4. ErbB receptor family has essential roles in mammalian development and homeostasis, and thus these receptors are considered as promising targets for anticancer drugs. 41, 42 Structural studies implicated the role of EGFR dimerization in the activation of cytoplasmic protein tyrosine kinase activity.43 Lemmon and co-workers demonstrated via TOXCAT assay that the C-terminal GXXXG motif was involved in homodimerization in ErbB proteins.44 Shai’s group revealed that the N-terminal GXXXG motif participates in the heterodimerization between ErbB1 and ErbB2.45 This conclusion was further verified by the ability of a synthetic ErbB1 transmembrane peptide to heterodimerize with ErbB2. Subsequent studies showed that the TMD peptide of ErbB1 and ErbB2 could specifically inhibit the auto-phosphorylation and subsequent signal transduction of their cognate receptors.46

2.3 Optical isomers of transmembrane helices Peptides composed of D-amino acids may retain their ability to interact with the binding partners of their naturally occurring enantiomers. Meanwhile, these peptides tend to be resistant to enzyme degradation, thus show desirable stability as potential anti-TMD probes (Figure 7).


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Figure 7. Targeting the TMDs with optical isomers. D-enantiomers possess same amino acid sequence but different residue orientations comparing with their L-counterpart, by changing the tilt angle between two helices, it is possible to bring the similar interacting surfaces orientation as the L-homodimer did and with some special stability and affinity to their TMD target.

This strategy has been successfully used to develop potential drugs against human immunodeficiency virus (HIV-1) infection. The N-terminal domain of HIV-1 glycoprotein 41 (gp41) was demonstrated to be involved in the infection of the host cell.47 Kliger et al. synthesized a 33-residue oligopeptide corresponding to the Nterminal domain of gp41, which showed inhibition of cell fusion through interaction with the N-terminus of gp41.48 ATR-FTIR spectroscopy demonstrated that two enantiomers have a similar secondary structure in lipid bilayers, suggesting that highly hydrophobic environment within TMDs may help to hide backbone polar groups and maintain the enantiomers’ secondary structure. Based on this result, Pritsker et.al showed that an all Damino acid peptide (D-WT) in line with the N-terminal domain of gp41 inhibited cell fusion through gp41.49 Interestingly, D-WT did not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein.


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Shai’s group synthesized D-amino acid peptides to target TMDs of glycophorin A (GPA) and T-cell receptor (TCR) complex. The synthesized all-D analog of GPA TMD was showed to form dimmer with native GPA TMD.50 In an alternative approach, a diastereomer of GPA TMD by replacing two L-valines with D-valines was synthesized. The designed peptide exhibited a dose-dependent dominant negative effect on the dimerization of GPA TMD in ToxR assay. Molecular dynamics showed that both the homodimer and heterodimer maintained a similar contact surface, which could explain why the D-GPA retained the ability to bind to L-GPA.51 Shai and co-workers also developed a D-peptidic probe inhibiting T-cell activation based on the peptide previously reported to interfere with the interaction between the TCR α-chain and the cluster of differentiation 3 (CD3) Tcell co-receptor.52 Similarly, both the all-D and the diastereomer analogs with an L-arginine residue or an Llysine residue were used to inhibit antigen-specific T-cell activation and adjuvant arthritis in vivo in rats. Importantly, compared with all-L forms, the diastereomer analogs showed greater immunosuppressive activity in vivo and in vitro, which could be attribute to its greater solubility and resistance to degradation.53, 54 In general, truncated peptides and optical isomers can be regarded as another type of modification on natural MP oligomer scaffolds. Similar to modification on small molecules, the success of such designed truncated peptides (or optical isomers) depends critically on the ability of modified peptides to retain the native interactions with their oligomer partners. With the help of molecular dynamics and MP structural information, peptide design is expected to become a more efficient and reliable method in modulating MP oligomers.

2.4 CHAMP peptides With the rapid development of technology, computational design has become an increasingly feasible method for the development of peptides targeting protein TMDs. Yin et al. developed a computer-based strategy, computed helical anti-membrane protein (CHAMP), to identify peptides that could sequence-specifically interact with TMD helices (Figure 8).55 The initial step in the development of the CHAMP peptidic probe involved the identification of a preferred TMD dimerization mode between the target and the CHAMP anti-TMD peptide based on the structures in the known TMD database. After threading the target sequence into one of the TMD helices, the CHAMP’s sequence was optimized using a Monte Carlo repacking algorithm. Finally, the noninteracting residues were selected for insertion and anchoring with an Ez potential model.56 The CHAMP ACS Paragon Plus Environment

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method was used to study two homologous integrins, αIIbβ3 and αVβ3, the activation of which is regulated by the heterodimeric TMD association. The activities of the designed CHAMP peptide in micelles, liposomes and mammalian cells were also confirmed in the study.55 In addition, Caputo et al. showed the direct interaction between the CHAMP peptide and the full-length αIIbβ3.57

Figure 8. Design of CHAMP peptides targeting TMDs. (A) The general procedure to predict CHAMP peptide interface: The first step is to select a backbone geometry from the known structures that contain motifs in TMD target. Then amino acid residues are added to backbone, followed by a side chain-repacking algorithm; (B) The sequence motifs of αIIbβ3 and αVβ3; (C) The predicted packing interface between the integrin and CHAMP; (D) Activation of integrins by CHAMP peptide. Modified from Reference 55. Figure 8 is reprinted with permission.

Natural peptides consist of α-amino acids, while Kritzer et al. designed β-peptides that were made of β-amino acids to inhibit protein-protein interactions.58 However, the computational design of β-peptides is more challenging due to the lack of appropriate force fields. Nonetheless, Shandler et al. successfully applied the CHAMP method to design a β-peptide that targeted the transmembrane helix of the integrin αIIbβ3.59 The secondary structure and orientation of the peptide in the phospholipid bilayer were characterized using circular dichroism spectroscopy and attenuated total internal reflectance IR spectroscopy. Furthermore, it was confirmed that the designed peptide could selectively interact with the isolated TMD of αIIbβ3 and activate the integrin in vitro by several other techniques including analytical ultracentrifugation, rupture force spectroscopy, and transmission electron microscopy. 59


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3. Screening of agents targeting TMDs 3.1 Traptamers Transmembrane peptide aptamers, also known as traptamers, are artificial transmembrane peptides that are selected for targeting specific TMD of MPs (Figure 9). The bovine papillomavirus E5 protein, a 44 amino acid oligopeptide, is the smallest oncoprotein.60 E5 transforms cells by interacting with the TMD of platelet-derived growth factor β receptor (PDGFβR) to initiate dimerization and trans-autophosphorylation.61 DiMaio’s group conjectured that the E5 protein could serve as a scaffold to facilitate the discovery of probes targeting other cellular MPs. They constructed a traptamer peptide library by replacing E5 TMD with random hydrophobic amino acid sequences. This library was successfully applied to screen for TMD peptides that recognize the human erythropoietin receptor (EpoR). An artificial oligopeptide that activated human EpoR (without affecting murine EpoR and PDGFβR) was identified.62 This result indicated that the sequence changes within the TMD of a viral oncogene product could benefit the recognition of a completely unrelated protein target. Using a similar strategy, the group developed traptamers to identify six 44 or 45 amino acid oligopeptides that recognized the TMD of CCR5, a co-receptor of human HIV.63


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Figure 9. Targeting the TMDs by traptmers. Traptmer library can be constructed by replacing the TMD of E5 peptide with hydrophobic residues. High affinity traptmers for the target MP TMD will be identified by genetic screening.

3.2 In silico screening High-throughput screening, which involves rapid screening of compound libraries, may provide a reasonable alternative to rational design when the molecular basis is unclear.2 Various screening methods have been used in discover molecules targeting MPs, including wet screening and in silico screening. Compared to wet screening, in silico screening allows a more cost-efficient approach to identify hit molecules from a large library, which has been widely used to identify small molecules targeting G-protein coupled receptor (GPCR), GLUTs and many other MP targets.


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A prominent example of in silico screening to identify novel GPCR ligands is PZM21, a selective µ-opioidreceptor (µOR) agonist with little side effects. Morphine is a widely used canonical opioid analgesic that exerts its effect by activating µOR signaling.64 However, the side-effects of severe respiratory depression caused by the activation of µOR downstream in the β-arrestin pathway, and other closely related receptors, limit its analgesic application.65-67 Manglik et al. docked over 3 million compounds from the ZINC database against the inactive form of the µOR structure. Structure-based optimization on high-affinity hits yielded PZM21 (IC50=1 nM), with a 1000-fold improvement in binding infinity. PZM21 shared no structural similarity with canonical opioid drugs, and exhibited different in vivo profiles, as it only modulated brain µOR with no obvious effect on spinal cord µOR. In vivo study demonstrated that PZM21 had high analgesic efficacy, and was less likely to produce respiratory depression and constipation side effects, which were some of the major limitations of canonical opioid agonists.68 This work highlighted structure-based screening and its ability to identify GPCR ligands with new chemotypes that offer new efficacy profiles. However, the authors pointed out that screening small molecules followed by extensive SAR studies on one subtype target to acquire high selectivity might not be applicable in all situations.68 Orphan receptors are formidable targets for drug discovery, mainly due to the difficulties involved in developing functional assays without known ligands.69 Huang et al. described a combined in silico screening and in vitro approach to study the ligands of these pharmacologically dark receptors.70 A non-selective GPR68 agonist, lorazepam, was first identified. Through iterative modeling and optimization of 3307 homology models, a putative binding mode of lorazepam-GPR68 was obtained, and further verified by site-mutation of the putative interacting residues within TMDs. Based on this optimized model, ogerin (a selective GPR68 ligand) was identified. Ogerin was suggested to interact with Glu180, a residue located in the TMD and was not involved in the lorazepam-GPR68 interaction, which contributed to the isoform selectivity. Using ogerin as a probe, some important biological functions of GPR68 were revealed in the mouse hippocampus. Two molecules that potentially bind to GPR65 were identified using the same method, which indicated its general applicability.70 Developing GLUTs inhibitors that directly block the uptake of glucose in cancer cells is another strategy that exploits the Warburg effect against cancers. The crystal structures of GLUT1,71 GLUT3,72 GLUT573 reveal that the GLUT family proteins have similar structures, with promising potential pockets for the development of ACS Paragon Plus Environment

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inhibitors. Thompson et al. performed virtual screening on an inward-open GLUT5 homology model and selected the 175 top-ranked compounds for in vitro tests.74 The hit compound, MSNBA, selectively inhibited GLUT5 without affecting GLUT1-4 activity. The docking and mutagenesis results suggested that the interaction between the amino group in MSNBA and His387 on GLUT5 determined the specificity of MSNBA towards GLUT5. This work implied that the subtle differences between the crystal structures of GLUTs could be exploited to develop selective inhibitors. Mishra et al. reported two GLUT4 selective inhibitors using in silico screening.75 They constructed a GLUT4 homology model and virtually screened 1.8 million compounds from the ZINC database. A cell-viability assay determined that two hit compounds were effective. Two hit compounds were observed to specifically inhibit GLUT4 (not GLUT1) mediated glucose uptake in HEK293 cells that exogenously express GLUT1 and GLUT4. The results of the docking the two hits to the GLUT1 crystal structure and GLUT4 homology model revealed the interacting residues that contribute to selectivity. This study highlighted the power of homology modeling in acquiring GLUT isoform selectivity. Whereas it is still challenging to accurately predict the molecular recognition toward different MP conformations and assess the off-target effects using in silico screening. With accumulating MP structural information and improved computational algorithm, in silico screening will continue to be an attractive strategy in identifying small molecules recognizing MP TMDs.

3.3 Wet screening Although examples are rare, wet screening can also serve as a powerful tool to identify small molecules regulating vital processes within TMDs. M. tuberculosis (MTB) is the causative agent of most cases of tuberculosis (TB). MTB can turn into a dormant state, thus nullifying the potency of many common antibiotics. Due to its vital role in energy metabolism that is required by both active and dormant bacteria, MTB ATPase has become an ideal drug target in the development of new anti-TB drugs.76 In 2005, Andries et al. identified a diarylquinoline-based drug, TMC207, also known as bedaquiline, using a whole-cell assay to inhibit multiple growth cycles of TB.77 Analysis of the resistant mutants identified TMC207, which acted on the TMD of the F0 subunit of mycobacterial ATP synthase. By blocking the ATPase rotation motor, TMC207 was able to inhibit both dormant and metabolizing cells.77 Biukovic et al. reported the structure of the MTB F1F0 ATP synthase ε ACS Paragon Plus Environment

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subunit and C-terminal segment, and presented a new model of the TMC207-ATPase binding mode. It was suggested that wedge-like TMC207 interacted with Trp15 and Phe50 in ring c and subunit ε, hindering the function of ring c’s rotation and subunit ε’s regulation of coupling, respectively.78 These studies offered more detailed information about the TMC207-ATPase binding mode, which led to the further discovery of TMC207 analogs that target resistant strains. In 2012, the FDA approved TMC207 for treating multidrug resistant TB.79 There are very few examples of using small molecule agents to target lateral transmembrane protein-protein interaction. Human herpes virus 4 (EBV) is a common virus in humans, and is related to several autoimmune diseases80 and B cell lymphomas.81 The latent membrane protein 1 (LMP-1) is an important oncoprotein involved in EBV infection. LPM-1 TMD5 forms a homo-trimeric complex to initiate constitutive signaling and immortalize B cells during EBV infection. Through alanine scanning, Yin and co-workers discovered that the fifth transmembrane helix of LMP-1 was responsible for homotrimerization. In addition, a mutation of Asp150 on the fifth transmembrane helix of LMP-1 abolished the expression of NF-κB, a downstream signaling factor of LMP-1. This residue was involved in the hydrogen bond network, which stabilizes the formation of LMP-1 oligomers.82 Yin and co-workers screened the NCI library, and identified an inhibitor (NSC 295242) to disrupt the LMP-1 TM5 interactions. NSC 295242 was found bound to the hot spot on TM5, and thus inhibited LMP-1 homotrimerization.83

4. Perspective Technological progress has made many previously undruggable targets feasible for therapeutic development. Nonetheless, the regulation of TMDs by small molecules or peptides is still considered a formidable task. Despite the great difficulty involved in the discovery of probes against TMDs, recent developments in both library screening and rational design have illuminated this obscure area. High-throughput screening, either target-based or phenotype-based, followed by an extensive SAR study, is always a useful approach to identify lead compounds. Aided by computational design together with improved structural knowledge obtained by cryo-EM, X-ray crystallography, top-down MS, and molecular dynamics, researchers have developed new procedures to obtain hits toward various MPs. In silico screening is becoming


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de rigueur for the discovery of lead compounds for TMDs, especially GPCRs. We expect that with more precise energy functions, in silico screening will become more accurate and efficient. As an alternative way to generate probes, rational design enables researchers to modify existing probes to improve their potency and stability, based on a comprehensive understanding of the underlying interaction modes. Rational design depends on a comprehensive understanding of MPs. The already discovered probes are a treasure trove, inspiring more novel and fascinating strategies for drugging the currently undruggable MP TMDs.


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5. Author Information Corresponding author *Email: [email protected] (H. Y.); [email protected] (S.Z.)

Funding Funding of this work was provided by National Natural Science Foundation of China (NSFC Grant No. 21572114) and Tsinghua University.

Notes The author declares no competing financial interest.


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6. Acknowledgements We thank the other group members for their comments and assistance on this work.


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7. Abbreviations MPs, membrane proteins; EGFR, epithelial growth factor receptor; TLRs, toll-like receptors; PRR, pattern recognition receptor; TMDs, transmembrane domains; LCA, lithocholic acid; FXR, Farnesoid X receptor; GLUTs, glucose transporters; Glc-Pts, glucose-platinum conjugates; Gal-Pts, galactose platinum conjugates; SAR, structure-activity relationship; LspA, lipoprotein signal peptidase II; CAT, chloramphenicol acetyltransferase; DN-TOXCAT, dominant-negative TOXCAT; IR, insulin receptor; LTA, lipoteichoic acid; LPS, lipopolysaccharide; RTK, receptor tyrosine kinase; gp41, HIV-1 glycoprotein 41; D-WT, D-amino acid peptide; GPA, glycophorin A; TCR, T-cell receptor; CD3, cluster of differentiation 3; CHAMP, computed helical anti-membrane protein; GPCR, G protein-coupled receptor; µOR, µ-opioid-receptor; PDGFβR, plateletderived growth factor β receptor; EpoR, erythropoietin receptor; MTB, M.tuberculosis; TB, tuberculosis; EBV, human herpesvirus 4; LMP-1, latent membrane protein 1.


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For Table of Contents Use Only Title: Small molecule and peptide recognition of protein transmembrane domains Authors: Xianfeng Zeng, Peiyao Wu, Chengbo Yao, Jiaqi Liang, Shuting Zhang, Hang Yin


Biochemistry

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Overviews of major methods to identify agents recognizing MP TMDs. 174x109mm (144 x 144 DPI)


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Biochemistry

Rational design approaches based on natural product scaffolds. One of the approaches for the discovery of new agents recognizing protein TMDs is to attach a new pharmacophore to natural small molecules (left). The high affinity between nature-existed molecules and their TMD targets would guide the new attached pharmacophore to the binding sites; Also, structural modifications on natural small molecules could improve the affinity between those natural product analogs and TMD target (right). 288x116mm (144 x 144 DPI)


Biochemistry

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The structure of selective TGR5 agonists 1-3. Compounds 1-3 were obtained by replacing hydroxyl group with amino group in scaffold of LCA. 154x77mm (144 x 144 DPI)


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Biochemistry

Warburg effect and strategies of developing small-molecule drugs exploiting Warburg effect. (A) Warburg effect: GLUTs are overexpressed in cancer cells to compensate for low metabolic efficiency, and enable unregulated cell growth; (B) Strategies exploiting Warburg effect: Glucose conjugates enable selective delivery of anticancer pharmacophores to the cancer cells (left); GLUTs inhibitors can directly block the glucose uptake in energy demanding cancer cells (right); (C) The structure of designed hexose conjugates and their aglycone moiety. 167x245mm (144 x 144 DPI)


Biochemistry

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The molecular structure of globomycin and its co-crystal structure of globomycin in complex with LspA. (A) The structure of globomycin (8) and its analog 9; (B) The crystal structure of LspA-globomycin complex. (PDB entry: 5DIR). Modified from Reference 20. Figure 5B is reprinted with permission. 313x116mm (144 x 144 DPI)


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Biochemistry

TOXCAT system and truncated peptides as probes recognizing MP TMDs. (A) TOXCAT system: MBP: maltose binding protein; TM1 and TM2: Two engineered transmembrane helices; TM1 and TM2 interact with each other and form a functional ToxR dimer. The ToxR dimer binds to ctx promoter and induces measurable synthesis of CAT. TOXCAT provides a general way to determine the heterodimerization of two transmembrane helices; (B) Truncated peptides: Once the interacting protein transmembrane helices are determined, the truncated TMDs of one oligo partner can be exploited to modulate native oligomerization. 209x176mm (144 x 144 DPI)


Biochemistry

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Targeting the TMDs with optical isomers. D-enantiomers possess same amino acid sequence but different residue orientations comparing with their L-counterpart, by changing the tilt angle between two helices, it is possible to bring the similar interacting surfaces orientation as the L-homodimer did and with some special stability and affinity to their TMD target. 268x183mm (144 x 144 DPI)


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Biochemistry

Design of CHAMP peptides targeting TMDs. (A) The general procedure to predict CHAMP peptide interface: The first step is to select a backbone geometry from the known structures that contain motifs in TMD target. Then amino acid residues are added to backbone, followed by a side chain-repacking algorithm; (B) The sequence motifs of αIIbβ3 and αVβ3; (C) The predicted packing interface between the integrin and CHAMP; (D) Activation of integrins by CHAMP peptide. Modified from Reference 55. Figure 8 is reprinted with permission. 181x89mm (144 x 144 DPI)


Biochemistry

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Targeting the TMDs by traptmers. Traptmer library can be constructed by replacing the TMD of E5 peptide with hydrophobic residues. High affinity traptmers for the target MP TMD will be identified by genetic screening. 120x130mm (220 x 220 DPI)


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Small Molecule and Peptide Recognition of Protein Transmembrane

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