Kapil Upadhyaya

Ubiquitin (Ub) cooperates with other post-translational modifications to provide a tiered opportunity for protein regulation. Deltex E3 ligases were previously implicated in ubiquitylation of ADP-ribose (ADPr)-containing macromolecules in vitro, generating a noncanonical mono-ADPr-Ub ester (MARUbe). We previously identified mono-ADPr ubiquitylation (MARUbylation) on PARP7 in cells, which was extended with K11-linked polyUb, suggesting an intricately regulated, multilayered post-translational modification. Here, we show that the Deltex DTX2 ubiquitylates ADPr modifications on PARP7 in cells, which depends on PARP7 catalytic activity. We further identify RNF114 as the E3 ligase responsible for K11-linked polyUb extension on sites of PARP7 MARUbylation. Using a chemoenzymatic approach, we developed a fluorescent Ub-ADPr probe and find that RNF114 explicitly recognizes MARUbylated species. We used AlphaFold3 to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identify a tandem Di19-UIM module in RNF114 as a MARUbe-binding domain (M-UBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we describe a small family of M-UBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call MARUbe-Targeted Ligases (M-UTLs).

Ubiquitin (Ub) cooperation with other post-translational modifications provides a tiered opportunity for protein regulation. Small modifications to Ub such as phosphorylation, acetylation, or ADP-ribosylation have varying impacts on signaling. The Deltex family of E3 ligases was previously implicated in the ubiquitylation of ADP-ribose (ADPr) and ADPr-containing macromolecules. Our previous work found ester-linked mono-ADPr ubiquitylation (MARUbylation) on PARP7 and PARP10 in cells and that this mark is extended with K11 polyUb. We previously screened for E3 ligases that interact with PARP7 through three different approaches and identified six candidates, including the Deltex family member DTX2. One of these hits, RNF114, interacts with various other PARPs, leading us to hypothesize that RNF114 binds to sites of MARUbylation and extends K11 polyUb. Here, we show that DTX2 generates the initial MARUbe on PARP7 in cells, which depends on PARP7 catalytic activity. The MARUbe on PARP7 is extended with K11 polyUb by RNF114. To investigate the mechanism of RNF114 reader/writer function, we developed a click chemistry-inspired chemoenzymatic approach to create a novel fluorescent Ub-ADPr probe for studying its interaction with RNF114. Strikingly, we found that RNF114 has a weak affinity for ADPr and Ub separately but explicitly recognizes the linkage between Ub and ADPr present in MARUbylated species. We used AlphaFold3 modeling to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identified a tandem Di19-UIM module in RNF114 as a M AR U be- b inding d omain (MUBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we described a small family of MUBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call M ARUbe- T argeted L igases (MUTLs).

We describe a formal synthesis of 10-aza-9-oxakalkitoxin, the hydroxalog of the cytotoxic marine natural product kalkitoxin, that features Mukaiyama Markovnikov silyl peroxidation of a terminal alkene and N–O bond formation as the central enabling steps.

A novel and efficient Cu(I)-catalyzed ligand- and base-free multipathway domino strategy has been developed for the synthesis of 2-substituted quinazolinones. The reaction utilizes 2-bromobenzamide and multiform substrates such as aldehydes, alcohols, and methyl arenes for a one-pot protocol, whereas TMSN₃ is used as a nitrogen source. A wide range of substrate scope, functional group tolerance, and operational simplicity are synthetically useful features.

A novel class of gallic acid based glycoconjugates were designed and synthesized as potential anticancer agents. Among all the compounds screened, compound 2a showed potent anticancer activity against breast cancer cells. The latter resulted in tubulin polymerization inhibition and induced G2/M cell cycle arrest, generation of reactive oxygen species, mitochondrial depolarization and subsequent apoptosis in breast cancer cells. In addition, ultraviolet-visible spectroscopy and fluorescence quenching studies of the compound with tubulin confirmed direct interaction of compounds with tubulin. Molecular modeling studies revealed that it binds at the colchicine binding site in tubulin. Further, 2a also exhibited potent in vivo anticancer activity in LA-7 syngeneic rat mammary tumor model. Current data projects its strong candidature to be developed as anticancer agent.

An efficient and simple strategy for the synthesis of a diverse range of anthraquinone-based aryl-C-glycosides has been developed. It involves the sequential Diels–Alder reaction and oxidative aromatization with the preformed glycosyl diene and dienophiles. The glycosyl dienes were obtained from simple sugars by tandem one-pot substitution and elimination reaction.