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{{Short description|Tool in chemical genetics}}
[[File:Bump-and-hole engineering.png|thumb|Schematic of bump-and-hole method.<ref name=":0" />]]
The '''bump-and-hole method''' is a tool in [[chemical genetics]] for studying a specific [[Protein isoform|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 complex|protein–ligand]] interface is engineered to achieve selectivity through [[Steric effects|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 (biochemistry)|cofactor]] of the target protein. Hole-modified proteins are [[Protein production|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.<ref name=":0">{{Cite journal|last=Islam|first=Kabirul|date=October 2018|title=The Bump-and-Hole Tactic: Expanding the Scope of Chemical Genetics|journal=Cell Chemical Biology|language=en|volume=25|issue=10|pages=1171–1184|doi=10.1016/j.chembiol.2018.07.001|pmc=6195450|pmid=30078633}}</ref>

== History ==
Inspiration for the bump-and-hole method was drawn from mutant [[Escherichia coli|''E. coli'']] strains that carried an A294S mutant version of phenylalanine [[Aminoacyl tRNA synthetase|tRNA synthetase]] and survived exposure to ''p''-fluoroPhe, which has a slight "bump" and is cytotoxic when incorporated during [[Translation (biology)|translation]]. The A294S mutant strain was able to incorporate Phe, but not the bumped ''p''-fluoroPhe due to steric crowding from the hydroxyl group of S294.<ref>{{Cite journal|last1=Kast|first1=Peter|last2=Hennecke|first2=Hauke|date=November 1991|title=Amino acid substrate specificity of ''Escherichia coli'' phenylalanyl-tRNA synthetase altered by distinct mutations|journal=Journal of Molecular Biology|language=en|volume=222|issue=1|pages=99–124|doi=10.1016/0022-2836(91)90740-W|pmid=1942071}}</ref> Conversely, a "hole" created by a T45G mutation in [[ribonuclease]] A expanded its substrate specificity from pyrimidine nucleobases to a larger purine, adenine.<ref>{{Cite journal|last1=delCardayré|first1=Stephen B.|last2=Raines|first2=Ronald T.|date=May 24, 1994|title=Structural determinants of enzymatic processivity|journal=Biochemistry|language=en|volume=33|issue=20|pages=6031–6037|doi=10.1021/bi00186a001|pmid=8193116}}</ref> Subsequent work in the laboratories 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.<ref>{{Cite journal|last1=Liu|first1=Chang C.|last2=Schultz|first2=Peter G.|date=2010-06-07|title=Adding New Chemistries to the Genetic Code|journal=Annual Review of Biochemistry|language=en|volume=79|issue=1|pages=413–444|doi=10.1146/annurev.biochem.052308.105824|pmid=20307192|issn=0066-4154}}</ref>

[[File:Bump-and-hole-11.png|alt=|thumb|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.<ref>{{Cite journal|last1=Belshaw|first1=Peter J.|last2=Schoepfer|first2=Joseph G.|last3=Liu|first3=Karen-Qianye|last4=Morrison|first4=Kim L.|last5=Schreiber|first5=Stuart L.|date=1995-10-16|title=Rational Design of Orthogonal Receptor–Ligand Combinations|journal=Angewandte Chemie International Edition in English|language=en|volume=34|issue=19|pages=2129–2132|doi=10.1002/anie.199521291|issn=0570-0833}}</ref>]]
The first bump-and-hole pair, developed by [[Stuart Schreiber]] and colleagues, was a bumped [[Ciclosporin|cyclosporin A]] small-molecule with an Ile replacing Val at position 11, and a hole-modified (S99T/F113A) [[cyclophilin]] mutant.<ref>{{Cite journal|last=Schreiber|first=Stuart L.|date=1998-08-01|title=Chemical genetics resulting from a passion for synthetic organic chemistry|url=http://www.sciencedirect.com/science/article/pii/S0968089698001266|journal=Bioorganic & Medicinal Chemistry|language=en|volume=6|issue=8|pages=1127–1152|doi=10.1016/S0968-0896(98)00126-6|pmid=9784856|issn=0968-0896|url-access=subscription}}</ref> Cyclosporin A is a [[Chemically induced dimerization|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 cell|T cells]] in a cell- and tissue-specific manner.<ref>{{Cite journal|last1=Belshaw|first1=Peter J.|last2=Schreiber|first2=Stuart L.|date=February 1997|title=Cell-Specific Calcineurin Inhibition by a Modified Cyclosporin|journal=Journal of the American Chemical Society|language=en|volume=119|issue=7|pages=1805–1806|doi=10.1021/ja9636146|issn=0002-7863}}</ref> More recently, this first bump-and-hole pair was used to induce the assembly of [[Ten-Eleven Translocation 2|ten-eleven translocation 2]] dioxygenase in cells for temporally controlled [[DNA demethylation]].<ref>{{Cite journal|last1=Lee|first1=Minjung|last2=Li|first2=Jia|last3=Liang|first3=Yi|last4=Ma|first4=Guolin|last5=Zhang|first5=Jixiang|last6=He|first6=Lian|last7=Liu|first7=Yuliang|last8=Li|first8=Qian|last9=Li|first9=Minyong|last10=Sun|first10=Deqiang|last11=Zhou|first11=Yubin|date=2017-04-05|title=Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling|journal=Journal of the American Chemical Society|language=en|volume=139|issue=13|pages=4659–4662|doi=10.1021/jacs.7b01459|issn=0002-7863|pmc=5385525|pmid=28294608}}</ref>

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

=== Kinases ===
[[File:Bump-and-hole-2.png|alt=|thumb|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.<ref name=":1" /> ]]
Human protein [[Kinase|kinases]] use [[Adenosine triphosphate|ATP]] as a cofactor to [[Phosphorylation|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|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.<ref name=":1">{{Cite journal|last1=Liu|first1=Yi|last2=Shah|first2=Kavita|last3=Yang|first3=Feng|last4=Witucki|first4=Laurie|last5=Shokat|first5=Kevan M.|date=February 1998|title=Engineering Src family protein kinases with unnatural nucleotide specificity|journal=Chemistry & Biology|language=en|volume=5|issue=2|pages=91–101|doi=10.1016/S1074-5521(98)90143-0|pmid=9495830|doi-access=free}}</ref> 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 [[Protein mass spectrometry|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 [[Cyclin-dependent kinase 1|CDK1]], Pho85, [[MAPK1|ERK2]], and JNK.<ref>{{Cite journal|last1=Ubersax|first1=Jeffrey A.|last2=Woodbury|first2=Erika L.|last3=Quang|first3=Phuong N.|last4=Paraz|first4=Maria|last5=Blethrow|first5=Justin D.|last6=Shah|first6=Kavita|last7=Shokat|first7=Kevan M.|last8=Morgan|first8=David O.|date=October 2003|title=Targets of the cyclin-dependent kinase Cdk1|url=http://www.nature.com/articles/nature02062|journal=Nature|language=en|volume=425|issue=6960|pages=859–864|doi=10.1038/nature02062|pmid=14574415|bibcode=2003Natur.425..859U|s2cid=4391711 |issn=0028-0836|url-access=subscription}}</ref>

Whereas 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.<ref>{{Cite journal|last1=Hertz|first1=Nicholas T.|last2=Berthet|first2=Amandine|last3=Sos|first3=Martin L.|last4=Thorn|first4=Kurt S.|last5=Burlingame|first5=Al L.|last6=Nakamura|first6=Ken|last7=Shokat|first7=Kevan M.|date=August 2013|title=A Neo-Substrate that Amplifies Catalytic Activity of Parkinson's-Disease-Related Kinase PINK1|journal=Cell|language=en|volume=154|issue=4|pages=737–747|doi=10.1016/j.cell.2013.07.030|pmc=3950538|pmid=23953109}}</ref>

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.<ref>{{Cite journal|last1=Bishop|first1=Anthony C.|last2=Shah|first2=Kavita|last3=Liu|first3=Yi|last4=Witucki|first4=Laurie|last5=Kung|first5=Chi-yun|last6=Shokat|first6=Kevan M.|date=February 1998|title=Design of allele-specific inhibitors to probe protein kinase signaling|journal=Current Biology|language=en|volume=8|issue=5|pages=257–266|doi=10.1016/S0960-9822(98)70198-8|pmid=9501066|doi-access=free}}</ref> 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.<ref>{{Cite journal|last1=Bishop|first1=Anthony C.|last2=Ubersax|first2=Jeffrey A.|last3=Petsch|first3=Dejah T.|last4=Matheos|first4=Dina P.|last5=Gray|first5=Nathanael S.|last6=Blethrow|first6=Justin|last7=Shimizu|first7=Eiji|last8=Tsien|first8=Joe Z.|last9=Schultz|first9=Peter G.|last10=Rose|first10=Mark D.|last11=Wood|first11=John L.|date=September 2000|title=A chemical switch for inhibitor-sensitive alleles of any protein kinase|url=http://www.nature.com/articles/35030148|journal=Nature|language=en|volume=407|issue=6802|pages=395–401|doi=10.1038/35030148|pmid=11014197|bibcode=2000Natur.407..395B|s2cid=4430890 |issn=0028-0836|url-access=subscription}}</ref>

=== BET proteins ===
[[File:Wikipic4.png|alt=|thumb|The bumped ET inhibitor has selectivity for L94A BET BD due to steric complementarity. The un-bumped I-BET inhibitor would promiscuously bind BDs.<ref name=":2" /> ]]
The [[Bromodomain|BET]] (Bromodomains and Extra Terminal) family of proteins contain conserved motifs known as bromodomains (BDs) responsible for recognizing [[Acetylation|acetylated]] lysine on nucleosomal [[Histone|histones.]]<ref>{{Cite journal|last1=Filippakopoulos|first1=Panagis|last2=Qi|first2=Jun|last3=Picaud|first3=Sarah|last4=Shen|first4=Yao|last5=Smith|first5=William B.|last6=Fedorov|first6=Oleg|last7=Morse|first7=Elizabeth M.|last8=Keates|first8=Tracey|last9=Hickman|first9=Tyler T.|last10=Felletar|first10=Ildiko|last11=Philpott|first11=Martin|date=December 2010|title=Selective inhibition of BET bromodomains|journal=Nature|language=en|volume=468|issue=7327|pages=1067–1073|doi=10.1038/nature09504|issn=0028-0836|pmc=3010259|pmid=20871596|bibcode=2010Natur.468.1067F}}</ref> Recently, four members of the BET family, BRD2, 3, 4, and BRDT, each containing two bromodomains, were identified as important regulators of transcription.<ref>{{Cite journal|last1=Shi|first1=Jian|last2=Wang|first2=Yifan|last3=Zeng|first3=Lei|last4=Wu|first4=Yadi|last5=Deng|first5=Jiong|last6=Zhang|first6=Qiang|last7=Lin|first7=Yiwei|last8=Li|first8=Junlin|last9=Kang|first9=Tiebang|last10=Tao|first10=Min|last11=Rusinova|first11=Elena|date=February 2014|title=Disrupting the Interaction of BRD4 with Diacetylated Twist Suppresses Tumorigenesis in Basal-like Breast Cancer|journal=Cancer Cell|language=en|volume=25|issue=2|pages=210–225|doi=10.1016/j.ccr.2014.01.028|pmc=4004960|pmid=24525235}}</ref> 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.<ref>{{Cite journal|last1=Filippakopoulos|first1=Panagis|last2=Qi|first2=Jun|last3=Picaud|first3=Sarah|last4=Shen|first4=Yao|last5=Smith|first5=William B.|last6=Fedorov|first6=Oleg|last7=Morse|first7=Elizabeth M.|last8=Keates|first8=Tracey|last9=Hickman|first9=Tyler T.|last10=Felletar|first10=Ildiko|last11=Philpott|first11=Martin|date=December 2010|title=Selective inhibition of BET bromodomains|journal=Nature|language=en|volume=468|issue=7327|pages=1067–1073|doi=10.1038/nature09504|issn=0028-0836|pmc=3010259|pmid=20871596|bibcode=2010Natur.468.1067F}}</ref> 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.<ref name=":2">{{Cite journal|last1=Baud|first1=M. G. J.|last2=Lin-Shiao|first2=E.|last3=Cardote|first3=T.|last4=Tallant|first4=C.|last5=Pschibul|first5=A.|last6=Chan|first6=K.-H.|last7=Zengerle|first7=M.|last8=Garcia|first8=J. R.|last9=Kwan|first9=T. T.- L.|last10=Ferguson|first10=F. M.|last11=Ciulli|first11=A.|date=2014-10-31|title=A bump-and-hole approach to engineer controlled selectivity of BET bromodomain chemical probes|journal=Science|language=en|volume=346|issue=6209|pages=638–641|doi=10.1126/science.1249830|issn=0036-8075|pmc=4458378|pmid=25323695|bibcode=2014Sci...346..638B}}</ref> 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 factor|transcription factors.]]<ref>{{Cite journal|last1=Runcie|first1=A. C.|last2=Zengerle|first2=M.|last3=Chan|first3=K.-H.|last4=Testa|first4=A.|last5=van Beurden|first5=L.|last6=Baud|first6=M. G. J.|last7=Epemolu|first7=O.|last8=Ellis|first8=L. C. J.|last9=Read|first9=K. D.|last10=Coulthard|first10=V.|last11=Brien|first11=A.|date=2018|title=Optimization of a "bump-and-hole" approach to allele-selective BET bromodomain inhibition|journal=Chemical Science|language=en|volume=9|issue=9|pages=2452–2468|doi=10.1039/C7SC02536J|issn=2041-6520|pmc=5909127|pmid=29732121}}</ref>

=== Glycosidases ===
[[File:Wikipic5.png|alt=|thumb|Schematic of bumped pro-drug and hole-modified enzyme, releasing the drug only in the presence of the bump-and-hole pair.<ref name=":3" />]]
[[Glycosidase|Glycosidases]] are a family of enzymes that catalyze the hydrolysis of [[Glycosidic bond|glycosidic bonds]]. These enzymes can cleave glycans from [[Glycosylation|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 [[Galactosidases|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 [[Prodrug|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 the 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.<ref name=":3">{{Cite journal|last1=Hou|first1=Jingli|last2=Pan|first2=Yiwa|last3=Zhu|first3=Dashuai|last4=Fan|first4=Yueyuan|last5=Feng|first5=Guowei|last6=Wei|first6=Yongzhen|last7=Wang|first7=He|last8=Qin|first8=Kang|last9=Zhao|first9=Tiechan|last10=Yang|first10=Qiang|last11=Zhu|first11=Yan|date=February 2019|title=Targeted delivery of nitric oxide via a 'bump-and-hole'-based enzyme–prodrug pair|url=http://www.nature.com/articles/s41589-018-0190-5|journal=Nature Chemical Biology|language=en|volume=15|issue=2|pages=151–160|doi=10.1038/s41589-018-0190-5|pmid=30598545|s2cid=58561892 |issn=1552-4450|url-access=subscription}}</ref>

=== ''N''-Acetylgalactosaminyl transferases ===
[[File:Wikipic6.png|alt=|thumb|Hole-modified BH GalNac-Ts paired with UDP-GalNac analogs to tag GalNac T substrates to be visualized with click chemistry.<ref name=":4" /> ]]
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 [[Glycoprotein|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 [[Uridine diphosphate|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 regard 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 the complexity of glycan elaboration in the secretory pathway.<ref name=":4">{{Cite journal|last1=Schumann|first1=Benjamin|last2=Malaker|first2=Stacy Alyse|last3=Wisnovsky|first3=Simon Peter|last4=Debets|first4=Marjoke Froukje|last5=Agbay|first5=Anthony John|last6=Fernandez|first6=Daniel|last7=Wagner|first7=Lauren Jan Sarbo|last8=Lin|first8=Liang|last9=Li|first9=Zhen|last10=Choi|first10=Junwon|last11=Fox|first11=Douglas Michael|date=April 2020|title=Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells|journal=Molecular Cell|volume=78 |issue=5 |language=en|pages=824–834.e15|doi=10.1016/j.molcel.2020.03.030 |pmc=7276986 |pmid=32325029|doi-access=free}}</ref>

== References ==
{{reflist}}

[[Category:Genetics]]
[[Category:Homology theory]]
[[Category:Human proteins]]

Bump and hole - История изменений

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