Rahul Kohli, M.D., Ph.D.
University of Pennsylvania
The Perelman School of Medicine
Department of Medicine
504 Johnson Pavilion
3610 Hamilton Walk
Philadelphia, PA 19104
Elucidating and exploiting the mechanism of natural product macrocycle biosynthesis to make improved antibiotics. Many natural product antibiotics are brought to their bioactive conformations by cyclization. Macrocyclization rigidifies the small molecules to make them more effective at binding to their molecular targets. In my graduate work, I examined the natural product assembly lines of non-ribosomal peptide synthetases. In this work, I helped to establish that, rather than simply being responsible for unloading of the synthetase modules, the C-terminal domain of these assembly lines can catalyze release and macrocyclization of the natural products ref (a). After this initial discovery, I showed that this mechanism is broad ranging across a wide range of natural products spanning anti-infective and anti-cancer agents. We leveraged these advances to develop a chemoenzymatic approach to access alternative classes of related natural products (ref (b)), target new therapeutic areas (ref (c)), or improve antibiotic activity or specificity (ref (d)). This work demonstrates my abilities to decipher complex enzymatic reaction mechanisms.
Trauger JW, Kohli RM, Mootz HD, Marahiel MA, Walsh CT (2000) Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase, Nature 407: 215-218.
Kohli RM, Burke MD, Tao J, Walsh CT (2003) Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules, J Am Chem Soc 125: 7160-7161.
Kohli RM, Takagi J, Walsh CT (2002) The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides, Proc Natl Acad Sci USA 99: 1247-1252 (PMC122175).
Kohli RM, Walsh CT, Burkart MD (2002) Biomimetic synthesis and optimization of cyclic peptide antibiotics, Nature 418: 658-661.
Targeting the SOS Pathway to combat antibiotic resistance. My clinical training has exposed me to the ever-increasing impact of antibiotic resistance and made it evident that “next generation” antibiotics only stall resistance. My involvement in making “next generation” antibiotics (see (1) above) made me realized the need to tackle the root cause, i.e., how antibiotic resistance arises. In this vein, we have focused on understanding how the DNA damage response of bacteria (also known as the SOS response) allows them to adapt to antibiotics or evolve and acquire resistance. Our detailed biochemical analyses (ref (a)) have allowed us to devise strategies for targeting the SOS response to potentiate antibiotics (ref (b)) and we have isolated first-in-class inhibitors of LexA (ref (c)). This work represents my lab’s effort to advance a new paradigm (ref (d)) in a field with great need for innovation.
Mo CY, Birdwell LD, Kohli RM (2014) Specificity Determinants for Autoproteolysis of LexA, a Key Regulator of Bacterial SOS Mutagenesis, Biochemistry 53:3158-68 (PMC4030785).
Mo CY, Manning SA, Roggiani M, Culyba MJ, Samuels AN, Sniegowski PD, Goulian M, Kohli RM (2016) Systematically Altering Bacterial SOS Activity under Stress Reveals Therapeutic Strategies for Potentiating Antibiotics, mSphere 1:e00163-16 (PMC4980697).
Mo CY, Culyba MJ, Selwood T, Kubiak JM, Hostetler ZM, Jurewicz AJ, Keller PM, Pope AJ, Quinn A, Schneck J, Widdowson KL, Kohli RM (2018) Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership, ACS Infect Dis 4:349-359 (PMC5893282).
Merrikh H, Kohli RM (2020) Targeting Evolution to Inhibit Antibiotic Resistance, FEBS J, 287: 4341-4352 (PMC7578009).
Explaining the basis for targeted mutagenesis by DNA deaminases. My clinical experiences have highlighted the importance of understanding how diversity arises on both sides of the host-pathogen interface. As a clear example of our ability to integrate nucleic acid chemistry with enzyme mechanisms, my lab has made great strides in understanding how targeted and purposeful mutation is used to improve our immune defenses. Activation Induced Deaminase (AID) is the key driver of antibody maturation, catalyzing the targeted deamination of cytosine to generate uracil within the immunoglobulin locus. We have helped to decipher how targeting takes place at the molecular level, demonstrating how particular hotspots in the genome are targeted by AID and its APOBEC3 relatives (refs (a) and (c)) and how the enzyme can discriminate DNA from RNA (ref (b)). Most recently, we have leveraged this knowledge to develop small molecule controllable genomic base editors. This work demonstrates my success in manipulating enzymes to reveal mechanism and alter function.
Kohli RM, Abrams SR, Gajula KS, Maul RW, Gearhart PJ, Stivers JT (2009) A portable hotspot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase, J Biol Chem 284: 22898-22904 (PMC2755697).
Nabel CS, Lee JW ,Wang LC Kohli RM (2013) Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase, Proc Natl Acad Sci USA 110: 14225–14230 (PMC3761612).
Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM (2014) High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase, Nucleic Acids Res 42: 9964-75 (PMC4150791).
Berríos KN, Evitt NH, DeWeerd RA, Ren D, Luo M, Barka A, Wang T, Bartman CR, Lan Y, Green AM, Shi J, Kohli RM (2021) Controllable genome editing with split-engineered base editors. Nat Chem Biol. 17:1262-1270.
Addressing the enigmatic mechanism of DNA demethylation. Despite the wealth of studies on the importance of 5-methylcytosine (mC) in mammalian genomes, the mechanism by which DNA can be demethylated has remained elusive. While high profile studies had suggested that deamination of mC or related analogs could be involved in DNA demethylation, the biochemical feasibility of this reaction had not been established. Further, with the discovery of TET family enzymes that can oxidize mC, new avenues for demethylation have been recently proposed. We were the first to show that enzymatic deamination of oxidized analogs of mC is a disfavored route for demethylation and that TDG can deplete 5-formylcytosine from genomes (ref (a)). We have also demonstrated that TET2 shows catalytic processivity (ref (b)), providing a mechanism for the generation of highly oxidized mC species despite the relative dearth of their precursors, uncovered new reactivities for TET enzymes (ref (c)), and helped to reveal new and unexpected DNA modifications which may mediate demethylation. Finally, we contributed to work led by Dr. Fraietta showing the clinical importance of TET2 in CAR-T therapy (ref (d)). This work demonstrates my role in helping to shape to the field’s current model for how methylation, deamination and oxidation collaborate to establish the epigenome.
Nabel CS, Jia H, Ye Y, Shen L, Goldschmidt HL, Stivers JT, Zhang Y, Kohli RM (2012) AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation, Nature Chem Biol 8:751-8 (PMC3427411).
Crawford DJ, Liu MY, Nabel CS, Cao XJ, Garcia BA, Kohli RM (2016) Tet2 Catalyzes Stepwise 5-Methylcytosine Oxidation by an Iterative and de novo Mechanism, J Am Chem Soc 138:730-3 (PMC4762542).
Ghanty U, Wang T, Kohli RM. (2020) Nucleobase Modifiers Identify TET Enzymes as Bifunctional DNA Dioxygenases Capable of Direct N-Demethylation, Angew Chem Int Ed, 59: 11312-11315 (PMC7332413).
Fraietta JA, Nobles CL, Sammons MA, Lundh S, Carty SA, Reich TJ, Cogdill AP, Morrissette JJD, DeNizio JE, Reddy S, Hwang Y, Gohil M, Kulikovskaya I, Nazimuddin F, Gupta M, Chen F, Everett JK, Alexander KA, Lin-Shiao E, Gee MH, Liu X, Young RM, Ambrose D, Wang Y, Xu J, Jordan MS, Marcucci KT, Levine BL, Garcia KC, Zhao Y, Kalos M, Porter DL, Kohli RM, Lacey SF, Berger SL, Bushman FD, June CH, Melenhorst JJ. (2018) Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells, Nature, 558: 307-312 (PMC 29849141).
Rational engineering of DNA modifying enzymes for epigenetic analysis and sequencing. Utilizing a structurally guided approach, we evolved the first TET variants that selectively stall oxidation at 5-hydroxymethylcytosine (hmC) (ref (a)). These modified enzymes offer a novel tool that is being applied to understand the role of 5-hydroxymethylcytosine, as distinct from the highly oxidized bases 5-formylcytosine and 5-carboxycytosine. In a similar vein, we have exploited our knowledge of APOBEC selectivity (ref (b)) to develop an enzymatic method to localize 5-hydroxymethylcytosine in genomic DNA (ref (c)). These advances demonstrate our success in harnessing structure-function insights into DNA modifying enzymes to apply them to reveal new biology.
Liu MY, Torabifard H, Crawford DJ, DeNizio JE, Cao XJ, Garcia BA, Cisneros GA, Kohli RM (2016) Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine. Nature Chem Biol, 3:181-187 (PMC5370579).
Schutsky EK, Nabel CS, Davis AKF, DeNizio JE, Kohli RM (2017) APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA. Nucleic Acids Res. 45:7655-7665 (PMC5570014).
Schutsky EK, DeNizio JE, Hu P, Liu MY, Nabel CS, Fabyanic EB, Hwang Y, Bushman FD, Wu H, Kohli RM. (2018) APOBEC-Coupled Epigenetic Sequencing permits low-input, bisulfite-free localization of 5-hydroxymethylcytosine at base resolution. Nature Biotech. 36: 1083–1090 (PMC6453757).
Caldwell BA, Liu MY, Prasasya RD, Wang T, DeNizio JE, Leu NA, Amoh NYA, Krapp C, Lan Y, Shields EJ, Bonasio R, Lengner CJ, Kohli RM, Bartolomei MS. (2021) Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency. Mol Cell. 81: 859-869 (PMC7897302).
While we conventionally think of genomic DNA as a simple polymer of A‘s, C‘s, G‘s, and T‘s, the chemistry of the genome is in fact far more interesting.
Our laboratory focuses on the DNA modifying enzymes that provide an added layer of complexity to the genome. These enzymes can be involved in the purposeful introduction of mutations or in the chemical modification of nucleobases, making DNA a remarkably dynamic entity. Many of these processes are at the heart of the battle between the immune system and pathogens or are central to epigenetics.
Our work can be broadly classified in two areas:
Enzymatic deamination, oxidation and methylation of cytosine bases, with a focus on AID/APOBEC DNA deaminases and TET oxygenases
Targeting Pathogen Pathways that Promote Evolution and Antibiotic Resistance, with a focus on the LexA/RecA axis governing the bacterial SOS response.
We utilize a broad array of approaches, which include 1) biochemical characterization of enzyme mechanisms, 2) chemical synthesis of enzyme probes, and 3) biological assays spanning bacteriology, immunology, and virology to study DNA modifying enzymes and pro-mutagenic pathways.
Our research program aims to understand diversity generating enzymes and pathways in vitro, to perturb their function in physiological settings, and to harness the biotechnological potential of these diversity-generating pathways.
|FIRST NAME||LAST NAME||TITLE|
|Jared||Parker||Senior Research Investigator||Jared.B.Parker@gmail.com|