Bump and hole

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Schematic of bump-and-hole method. Bump-and-hole engineering.png
Schematic of bump-and-hole method.

The bump-and-hole method is a tool in chemical genetics for studying a specific isoform in a protein family without perturbing the other members of the family. The unattainability of isoform-selective inhibition due to structural homology in protein families is a major challenge of chemical genetics. With the bump-and-hole approach, a protein–ligand interface is engineered to achieve selectivity through steric complementarity while maintaining biochemical competence and orthogonality to the wild type pair. Typically, a "bumped" ligand/inhibitor analog is designed to bind a corresponding "hole-modified" protein. Bumped ligands are commonly bulkier derivatives of a cofactor of the target protein. Hole-modified proteins are recombinantly expressed with an amino acid substitution from a larger to smaller residue, e.g. glycine or alanine, at the cofactor binding site. The designed ligand/inhibitor has specificity for the engineered protein due to steric complementarity, but not the native counterpart due to steric interference. [1]

Contents

History

Inspiration for the bump-and-hole method was drawn from mutant E. coli strains which carried an A294S mutant version of phenylalanine tRNA synthetase and survived exposure to p-FluoroPhe, a slightly bumped phenylalanine analog which is cytotoxic when incorporated in translation. The A294S mutant strain was able to incorporate Phe, but not the bumped p-FluoroPhe due to steric crowding from the hydroxymethylene of S294. [2] Later work in the labs of Peter G. Schultz and David A. Tirrell showed that a hole-modified A294G phenylalanine tRNA synthetase mutant was able to incorporate the bumped p-FluoroPhe in translation, demonstrating that steric manipulation can successfully broaden substrate scope, even for the highly specific aminoacyl synthetase. [3]

The first reported bump-and-hole pair. Hole-modified S99T/F113A mutant cyclophilin has an expanded hydrophobic pocket to accept a methyl bump in cyclosporin A analog MeIle11CsA. Bump-and-hole-11.png
The first reported bump-and-hole pair. Hole-modified S99T/F113A mutant cyclophilin has an expanded hydrophobic pocket to accept a methyl bump in cyclosporin A analog MeIle11CsA.

The first bump-and-hole pair, developed by Stuart Schreiber and colleagues, was a bumped cyclosporin A small-molecule with an Ile replacing Val at position 11, and a hole-modified (S99T/F113A) cyclophilin mutant. [5] Cyclosporin A is a chemical inducer of dimerization (CID) of cyclophilin. This first bump-and-hole pair was engineered to improve the binding efficiency between wild type cyclosporin A and cyclophilin, thereby giving more efficient CID. The bumped cyclosporin A was found to interact efficiently with the hole-modified cyclophilin mutant, but not endogenous cyclophilin. The orthogonal CID pair was used to inhibit calcineurin-mediated dephosphorylation of nuclear factor of activated T cells in a cell- and tissue-specific manner. [6] More recently, this first bump-and-hole pair was used to induce the assembly of ten-eleven translocation 2 dioxygenase in cells for temporally controlled DNA demethylation. [7]


Applications

As structural information about protein-ligand interfaces have become available, bump-and-hole pairs have been used to elucidate the substrates of specific proteins from various protein classes, as well as develop orthogonal neoenzyme-neosubstrate therapeutics.

Kinases

The bumped ATP analog N6-cyclopentyl ATP cannot bind wild type v-Src kinase, but can bind its bump-and-hole pair, I338G v-Src kinase. Bump-and-hole-2.png
The bumped ATP analog N6-cyclopentyl ATP cannot bind wild type v-Src kinase, but can bind its bump-and-hole pair, I338G v-Src kinase.

Human protein kinases use ATP as a cofactor to phosphorylate substrate proteins. Kinases play critical roles in complex cell signaling networks. Conserved ATP binding sites and similar catalytic mechanisms pose a challenge to selectively inhibiting a particular kinase to determine its function. Kevan Shokat's lab has developed bump-and-hole pairs using kinase mutants with bulky "gatekeeper" residues in the ATP-binding pocket replaced by Gly or Ala, and bulky ATP analogs. In early work, v-Src kinase I338A/G mutants were shown to accept [γ-32P]-labeled bumped N6-cyclopentyl and N6-benzyl ATP analogs as alternative cofactors to radiolabel its substrates. [8] Only the mutant kinase was able to bind the bumped ATP analogs, allowing labeling of substrates specific to the engineered v-Src kinase. Purification and MS-based proteomics yielded the substrates of v-Src kinase. Hole-modified kinase and bumped ATP analog pairs enabled substrate profiling of several other kinases, including CDK1, Pho85, ERK2, and JNK. [9]

While bumped ATM analogs can help deconvolute kinase substrate profiles, one drawback of this strategy is the cell impermeability of the bumped analogs. To get around this, the Shokat group demonstrated that a bumped ATP analog, kinetin ATP or KTP, could be synthesized endogenously in cells cultured with kinetin. Once synthesized, it can activate a PINK1 kinase mutant, which is otherwise inactive in the absence of the bumped analog. Inactive PINK1 is implicated in Parkinson's disease (PD). In the context of PD, the mutant PINK1-KTP pair represents an orthogonal neoenzyme-neosubstrate therapeutic. [10]

The Shokat group also applied the bump-and-hole approach to develop selective, cell-permeable bumped inhibitors of mutant kinases. For the I338G v-Src kinase, a 4-amino-l-tert-butyl-3-(p-methylphenyl)pyrazolo[3,4-d]pyrimidine (PP1) derivative called p-tButPhe-PP1 was developed for selective inhibition; steric bulk precluded binding to the wild type v-Src kinase. In mammalian cell lines, active v-Src kinase is required for transformation by Rous sarcoma virus. In cell lines expressing I338G v-Src kinase and transfected with RSV, treatment with p-tButPhe-PP1 caused the reversal of transformation, suggesting inhibition of the kinase mutant. [11] Later, the group developed bumped inhibitors 1-naphthyl PP1 (NA-PP1) and 1-methylnaphthyl PP1 (MN-PP1), which inhibited hole-modified yeast kinases with IC50 values in low nanomolar concentrations. [12]

BET proteins

The bumped ET inhibitor has selectivity for L94A BET BD due to steric complementarity. The un-bumped I-BET inhibitor would promiscuously bind BDs. Wikipic4.png
The bumped ET inhibitor has selectivity for L94A BET BD due to steric complementarity. The un-bumped I-BET inhibitor would promiscuously bind BDs.

The BET (Bromodomains and Extra Terminal) family of proteins contain conserved motifs known as bromodomains (BDs) responsible for recognizing acetylated lysine on nucleosomal histones. [14] Recently, four members of the BET family, BRD2, 3, 4, and BRDT, each containing two bromodomains, were identified as important regulators of transcription. [15] In order to probe bromodomain-specific functions of members of the BET family, small-molecule inhibitors JQ1 and I-BET were developed, but they lacked inter- and intra-BET (between BDs on the same protein) selectivity. [16] The lab of Alessio Ciulli produced bump-and-hole pairs consisting of ET, a derivative of I-BET with an ethyl bump, and different members of the BET family with an L94A mutation in their BD1. [13] ET was found to have a 160-fold greater specificity for hole-modified BD1 of BET mutants compared to compared to the BDs of wild type BET proteins, giving BD-specific inhibition. The BD-ET bump-and-hole pairs were used to show that selective inhibition of BD1 in a BET protein disrupts chromatin engagement. Recently, the Ciulli group developed a new bump-and-hole pair consisting of BET mutants with a Leu to Val mutation in a BD and the bumped small-molecule inhibitor 9-ME-1. This bumped inhibitor was found to have an IC50 of 200nM and over 100-fold specificity for the L/V BET mutant BD over wild type BDs. This bump-and-hole pair allowed selective inhibition of specific BDs in specific BET proteins, elucidating their role in human cells. It was found that while BD1 is important for chromatin localization of BET proteins, BD2 regulates gene expression by binding and recruiting non-histone acetylated proteins, such as transcription factors. [17]

Glycosidases

Schematic of bumped pro-drug and hole-modified enzyme, releasing the drug only in the presence of the bump-and-hole pair. Wikipic5.png
Schematic of bumped pro-drug and hole-modified enzyme, releasing the drug only in the presence of the bump-and-hole pair.

Glycosidases are a family of enzymes that catalyzes the hydrolysis of glycosidic bonds. These enzymes can cleave glycans from glycosylated proteins, one of the most common forms of post-translational modification. In a recent therapeutic application of the bump-and-hole method, a hole-modified galactosidase was paired with a bumped galactosyl-pro-drug. Jingli Hou and colleagues sought to deliver nitric oxide, an important messenger for promoting tissue growth processes like angiogenesis and vasculogenesis, in a spatiotemporally controlled manner. They opted for a pro-drug system, wherein the NO-releasing drug, NONOate, is initially glycosylated. Once the glycosylated NONOate enters cells and is exposed to glycosidases, NO is released. However, non-tissue-specific systemic release of NO, which can reduce therapeutic efficiency and cause harmful side effects, from these pro-drugs was evident due to widespread distribution of endogenous glycosidases. To get around this, Hou et al. developed a bumped pro-drug via methylation of the O6 of the galactose moiety of galactosyl-NONOate. They engineered a corresponding hole-modified β-galactosidase mutant, A4-β-GalH363A with specificity for the bumped galactosyl-NONOate. The bumped pro-drug evaded cleavage by wild type β-galactosidase due to the methylated O6 of the galactose moiety and strict regioselectivity of glycosidases. NO was released in tissues only in the presence of both the bumped galactosyl-NONOate and the hole-modified β-galactosidase mutant, giving spatiotemporal control of delivery. Hou et al. found markedly increased therapeutic efficiency of NO delivery via the bump-and-hole engineered system, compared to the unmodified pro-drug, in rat hindlimb ischemia and mouse acute kidney injury models. [18]

N-Acetylgalactosaminyl transferases

Hole-modified BH GalNac-Ts paired with UDP-GalNac analogs to tag GalNac T substrates to be visualized with click chemistry. Wikipic6.png
Hole-modified BH GalNac-Ts paired with UDP-GalNac analogs to tag GalNac T substrates to be visualized with click chemistry.

The N-Acetylgalactosaminyl transferase (GalNac Ts) family transfers N-Acetylgalactosamine to the Ser/Thr side chains (O-linked glycosylation) of its substrates, using UDP-GalNac as a cofactor. Like kinases, substrate profiling for specific isoforms of GalNac Ts has been difficult to achieve. The absence of a glycosylation consensus sequence and the variability of glycan elaboration pose a challenge to studying O-GalNac glycoproteins. Further, GalNac transferase knockout strategies are ineffective because the activity of isoforms in the family is both redundant and competitive, such that compensation occurs upon KO. Recently, Schumann et al. applied the bump-and-hole strategy to engineer bumped alkyne-containing UDP-GalNac analogs and double hole-modified I253A/L310A mutant GalNac Ts (BH GalNac Ts). The UDP-alkyne analogs were specific to complementary BH GalNac Ts, which were shown to maintain the biochemical competence of wild type GalNac Ts, with regards to structure, localization, and substrate specificity. This bump-and-hole pair attached a bio-orthogonal label, visualizable through click chemistry, on the substrates of distinct GalNac T isoforms, deconvolving substrate profiles while displaying complexity of glycan elaboration in the secretory pathway. [19]

Related Research Articles

A protein phosphatase is a phosphatase enzyme that removes a phosphate group from the phosphorylated amino acid residue of its substrate protein. Protein phosphorylation is one of the most common forms of reversible protein posttranslational modification (PTM), with up to 30% of all proteins being phosphorylated at any given time. Protein kinases (PKs) are the effectors of phosphorylation and catalyse the transfer of a γ-phosphate from ATP to specific amino acids on proteins. Several hundred PKs exist in mammals and are classified into distinct super-families. Proteins are phosphorylated predominantly on Ser, Thr and Tyr residues, which account for 79.3, 16.9 and 3.8% respectively of the phosphoproteome, at least in mammals. In contrast, protein phosphatases (PPs) are the primary effectors of dephosphorylation and can be grouped into three main classes based on sequence, structure and catalytic function. The largest class of PPs is the phosphoprotein phosphatase (PPP) family comprising PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7, and the protein phosphatase Mg2+- or Mn2+-dependent (PPM) family, composed primarily of PP2C. The protein Tyr phosphatase (PTP) super-family forms the second group, and the aspartate-based protein phosphatases the third. The protein pseudophosphatases form part of the larger phosphatase family, and in most cases are thought to be catalytically inert, instead functioning as phosphate-binding proteins, integrators of signalling or subcellular traps. Examples of membrane-spanning protein phosphatases containing both active (phosphatase) and inactive (pseudophosphatase) domains linked in tandem are known, conceptually similar to the kinase and pseudokinase domain polypeptide structure of the JAK pseudokinases. A complete comparative analysis of human phosphatases and pseudophosphatases has been completed by Manning and colleagues, forming a companion piece to the ground-breaking analysis of the human kinome, which encodes the complete set of ~536 human protein kinases.

<span class="mw-page-title-main">Tyrosine kinase</span> Class hi residues

A tyrosine kinase is an enzyme that can transfer a phosphate group from ATP to the tyrosine residues of specific proteins inside a cell. It functions as an "on" or "off" switch in many cellular functions.

<span class="mw-page-title-main">Cyclin-dependent kinase</span> Class of enzymes

Cyclin-dependent kinases (CDKs) are a predominant group of serine/threonine protein kinases involved in the regulation of the cell cycle and its progression, ensuring the integrity and functionality of cellular machinery. These regulatory enzymes play a crucial role in the regulation of eukaryotic cell cycle and transcription, as well as DNA repair, metabolism, and epigenetic regulation, in response to several extracellular and intracellular signals. They are present in all known eukaryotes, and their regulatory function in the cell cycle has been evolutionarily conserved. The catalytic activities of CDKs are regulated by interactions with CDK inhibitors (CKIs) and regulatory subunits known as cyclins. Cyclins have no enzymatic activity themselves, but they become active once they bind to CDKs. Without cyclin, CDK is less active than in the cyclin-CDK heterodimer complex. CDKs phosphorylate proteins on serine (S) or threonine (T) residues. The specificity of CDKs for their substrates is defined by the S/T-P-X-K/R sequence, where S/T is the phosphorylation site, P is proline, X is any amino acid, and the sequence ends with lysine (K) or arginine (R). This motif ensures CDKs accurately target and modify proteins, crucial for regulating cell cycle and other functions. Deregulation of the CDK activity is linked to various pathologies, including cancer, neurodegenerative diseases, and stroke.

<span class="mw-page-title-main">Chemical biology</span> Scientific discipline

Chemical biology is a scientific discipline between the fields of chemistry and biology. The discipline involves the application of chemical techniques, analysis, and often small molecules produced through synthetic chemistry, to the study and manipulation of biological systems. Although often confused with biochemistry, which studies the chemistry of biomolecules and regulation of biochemical pathways within and between cells, chemical biology remains distinct by focusing on the application of chemical tools to address biological questions.

<span class="mw-page-title-main">Bromodomain</span>

A bromodomain is an approximately 110 amino acid protein domain that recognizes acetylated lysine residues, such as those on the N-terminal tails of histones. Bromodomains, as the "readers" of lysine acetylation, are responsible in transducing the signal carried by acetylated lysine residues and translating it into various normal or abnormal phenotypes. Their affinity is higher for regions where multiple acetylation sites exist in proximity. This recognition is often a prerequisite for protein-histone association and chromatin remodeling. The domain itself adopts an all-α protein fold, a bundle of four alpha helices each separated by loop regions of variable lengths that form a hydrophobic pocket that recognizes the acetyl lysine.

<span class="mw-page-title-main">Enzyme inhibitor</span> Molecule that blocks enzyme activity

An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a specific chemical reaction by binding the substrate to its active site, a specialized area on the enzyme that accelerates the most difficult step of the reaction.

<span class="mw-page-title-main">BRAF (gene)</span> Protein-coding gene in the species Homo sapiens

BRAF is a human gene that encodes a protein called B-Raf. The gene is also referred to as proto-oncogene B-Raf and v-Raf murine sarcoma viral oncogene homolog B, while the protein is more formally known as serine/threonine-protein kinase B-Raf.

A non-receptor tyrosine kinase (nRTK) is a cytosolic enzyme that is responsible for catalysing the transfer of a phosphate group from a nucleoside triphosphate donor, such as ATP, to tyrosine residues in proteins. Non-receptor tyrosine kinases are a subgroup of protein family tyrosine kinases, enzymes that can transfer the phosphate group from ATP to a tyrosine residue of a protein (phosphorylation). These enzymes regulate many cellular functions by switching on or switching off other enzymes in a cell.

<span class="mw-page-title-main">SU6656</span> Chemical compound

SU6656 is a Src family kinase inhibitor developed by the biotechnology company SUGEN Inc in 2000. SU6656 was initially identified as a Src kinase inhibitor by virtue of its ability to reverse an effect that an activated mutant form of Src has on the actin cytoskeleton, namely the formation of podosome rosettes, otherwise known as invadopodia. SU6656 was initially published as a Src family kinase inhibitor with selectivity relative to Platelet-derived growth factor receptor Tyrosine kinase. Subsequent studies have confirmed that SU6656 is relatively selective for Src family kinases, but some additional biochemical activities have been identified including: BRSK2, AMPK, Aurora C, Aurora B, CaMKKβ. The inhibition of these kinases in biochemical reactions in vitro does not necessarily indicate that these kinases are targets of SU6656 in cells.

Bcr-Abl tyrosine-kinase inhibitors (TKI) are the first-line therapy for most patients with chronic myelogenous leukemia (CML). More than 90% of CML cases are caused by a chromosomal abnormality that results in the formation of a so-called Philadelphia chromosome. This abnormality was discovered by Peter Nowell in 1960 and is a consequence of fusion between the Abelson (Abl) tyrosine kinase gene at chromosome 9 and the break point cluster (Bcr) gene at chromosome 22, resulting in a chimeric oncogene (Bcr-Abl) and a constitutively active Bcr-Abl tyrosine kinase that has been implicated in the pathogenesis of CML. Compounds have been developed to selectively inhibit the tyrosine kinase.

<span class="mw-page-title-main">BIM-1</span> Biological protein kinase C inhibitor

BIM-1 and the related compounds BIM-2, BIM-3, and BIM-8 are bisindolylmaleimide-based protein kinase C (PKC) inhibitors. These inhibitors also inhibit PDK1 explaining the higher inhibitory potential of LY33331 compared to the other BIM compounds a bisindolylmaleimide inhibitor toward PDK1.

<span class="mw-page-title-main">Balanol</span> Fungal metabolite

Balanol is a fungal metabolite produced by the fungus Verticillium balanoides. It is a potent inhibitor of the serine/threonine kinases protein kinase A (PKA) and protein kinase C (PKC), binding in a similar manner with that of ATP. Balanol was discovered in 1993 in the search for novel inhibitors of PKC, a member of a family of serine/threonine kinases whose overactivation is associated with numerous human diseases of signal transduction including cancer. However, much of the research on balanol focuses on how chemical modifications of the molecular structure affect binding to PKA. Indeed, balanol, its chemically altered analogs, and their interactions with PKA in particular are used to illuminate the roles of selectivity and protein flexibility in the inhibition of kinases. For instance, the X-ray crystal structure of balanol in complex with PKA was used in order to confer selectivity and to improve pharmacological efficacy of inhibitors of the H. sapiens Akt (PKB), another serine/threonine protein kinase implicated in the proper functioning of many cellular processes.

c-Met inhibitors are a class of small molecules that inhibit the enzymatic activity of the c-Met tyrosine kinase, the receptor of hepatocyte growth factor/scatter factor (HGF/SF). These inhibitors may have therapeutic application in the treatment of various types of cancers.

<span class="mw-page-title-main">JQ1</span> Chemical compound

JQ1 is a thienotriazolodiazepine and a potent inhibitor of the BET family of bromodomain proteins which include BRD2, BRD3, BRD4, and the testis-specific protein BRDT in mammals. BET inhibitors structurally similar to JQ1 are being tested in clinical trials for a variety of cancers including NUT midline carcinoma. It was developed by the James Bradner laboratory at Brigham and Women's Hospital and named after chemist Jun Qi. The chemical structure was inspired by patent of similar BET inhibitors by Mitsubishi Tanabe Pharma [WO/2009/084693]. Structurally it is related to benzodiazepines. While widely used in laboratory applications, JQ1 is not itself being used in human clinical trials because it has a short half life.

mTOR inhibitors Class of pharmaceutical drugs

mTOR inhibitors are a class of drugs that inhibit the mammalian target of rapamycin (mTOR), which is a serine/threonine-specific protein kinase that belongs to the family of phosphatidylinositol-3 kinase (PI3K) related kinases (PIKKs). mTOR regulates cellular metabolism, growth, and proliferation by forming and signaling through two protein complexes, mTORC1 and mTORC2. The most established mTOR inhibitors are so-called rapalogs, which have shown tumor responses in clinical trials against various tumor types.

<span class="mw-page-title-main">Src inhibitor</span>

Src inhibitor is a class of inhibitors that targets the Src kinase family of tyrosine kinase, which is transcribed by the Src proto-oncogene that potentially induce malignant transformations of certain cells. Because of the crucial position of the Src kinase in cells, Src inhibitors are potential antineoplastic agents for e.g. pancreatic cancer, breast cancer and stomach cancer

<i>O</i>-GlcNAc

O-GlcNAc is a reversible enzymatic post-translational modification that is found on serine and threonine residues of nucleocytoplasmic proteins. The modification is characterized by a β-glycosidic bond between the hydroxyl group of serine or threonine side chains and N-acetylglucosamine (GlcNAc). O-GlcNAc differs from other forms of protein glycosylation: (i) O-GlcNAc is not elongated or modified to form more complex glycan structures, (ii) O-GlcNAc is almost exclusively found on nuclear and cytoplasmic proteins rather than membrane proteins and secretory proteins, and (iii) O-GlcNAc is a highly dynamic modification that turns over more rapidly than the proteins which it modifies. O-GlcNAc is conserved across metazoans.

BET inhibitors are a class of drugs that reversibly bind the bromodomains of Bromodomain and Extra-Terminal motif (BET) proteins BRD2, BRD3, BRD4, and BRDT, and prevent protein-protein interaction between BET proteins and acetylated histones and transcription factors.

<span class="mw-page-title-main">Targeted covalent inhibitors</span>

Targeted covalent inhibitors (TCIs) or Targeted covalent drugs are rationally designed inhibitors that bind and then bond to their target proteins. These inhibitors possess a bond-forming functional group of low chemical reactivity that, following binding to the target protein, is positioned to react rapidly with a proximate nucleophilic residue at the target site to form a bond.

<span class="mw-page-title-main">Orthogonal ligand-protein pair</span>

Orthogonal ligand-protein pairs are a protein-ligand binding pair made to be independent of the original binding pair. This is done by taking a mutant protein, which is activated by a different ligand. The intention here is that the orthogonal ligand will not interact with the original protein. The original protein will also be designed to not interact with the orthogonal ligand in certain cases.

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  19. 1 2 Schumann, Benjamin; Malaker, Stacy Alyse; Wisnovsky, Simon Peter; Debets, Marjoke Froukje; Agbay, Anthony John; Fernandez, Daniel; Wagner, Lauren Jan Sarbo; Lin, Liang; Li, Zhen; Choi, Junwon; Fox, Douglas Michael (April 2020). "Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells". Molecular Cell. 78 (5): 824–834.e15. doi: 10.1016/j.molcel.2020.03.030 . PMC   7276986 . PMID   32325029.
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