Hydroxylammonium derivatives for selective active-site lysine modification in the anti-virulence bacterial target DHQ1 enzyme

María Maneiro, Emilio Lence, Marta Sanz-Gaiterob, José M. Otero, Mark J. van Raaij, Paul Thompson, Alastair R. Hawkins and Concepción González-Bello

Org. Chem. Front., 2019
DOI: 10.1039/C9QO00453J 

Targeted irreversible inhibitors bearing electrophiles that become activated towards covalent bond formation upon binding to a specific protein/enzyme is an emerging area in drug discovery. Targeting lysine residues is challenging due to the intrinsically low reactivity of the amino group at physiological pH. Herein we report the first example of a hydroxylammonium derivative that causes a specific covalent modification of an active-site and a sterically inaccessible lysine residue of an enzyme. The described ligands, compounds 1–3, were rationally designed to be activated towards covalent bond formation upon binding to the type I dehydroquinase (DHQ1) enzyme for the development of new anti-virulence agents to combat the widespread resistance to antibiotics. Evidence in atomic detail for the covalent modifications caused by the ligands to the catalytic Lys170 by the formation of a stable secondary amine is provided by the resolution at 1.08–1.25 Å of the crystal structures of DHQ1 from Salmonella typhi enzyme adducts. In addition, the first crystal structure of the addition intermediate adduct at 1.4 Å of a Schiff base formation reaction by using an analog of the natural substrate, compound 4, is also reported. Molecular dynamics simulation studies on non-covalent enzyme/ligand complexes and a two-dimensional QM/MM umbrella sampling simulation study suggested that a direct displacement by Lys170 with the release of NH2OH would be feasible. These studies might open up new opportunities for the development of novel lysine-targeted irreversible inhibitors bearing a methylhydroxylammonium moiety as a latent electrophile.

Targeted Covalent Inhibition of Telomerase

Rick Betori, Yue Liu, Ding Wu, Rama Mishra, Scott Cohen, Stephen J. Kron, Karl Scheidt
ChemRxiv, 2019
doi: 10.26434/chemrxiv.8977457.v1

Telomerase is a ribonuceloprotein complex responsible for maintaining telomeres and protecting chromosomal integrity. The human telomerase reverse transcriptase (hTERT) is expressed in ~90% of cancer cells where it confers the capacity for limitless proliferation. Along with its established role in telomere lengthening, telomerase also serves non-canonical extra-telomeric roles in oncogenic signaling, resistance to apoptosis, and enhanced DNA damage response. Here, we report a new class of natural product-inspired covalent inhibitors of telomerase that target the catalytic active site. We developed rationally designed probe compounds that modulate both the telomeric and extra-telomeric activities of telomerase and present new opportunities to investigate the diverse functions of this unique molecular machine.

Enhancement of the Anti-Aggregation Activity of a Molecular Chaperone Using a Rationally Designed Post-Translational Modification

Philip R. Lindstedt, Francesco A. Aprile, Maria J. Matos, Michele Perni, Jean B. Bertoldo, Barbara Bernardim, Quentin Peter, Gonzalo Jiménez-Osés, Tuomas P. J. Knowles, Christopher M. Dobson, Francisco Corzana, Michele Vendruscolo, and Gonçalo J. L. Bernardes

ACS Cent. Sci. 2019

doi: 10.1021/acscentsci.9b00467

Protein behavior is closely regulated by a plethora of post-translational modifications (PTMs). It is therefore desirable to develop approaches to design rational PTMs to modulate specific protein functions. Here, we report one such method, and we illustrate its successful implementation by potentiating the anti-aggregation activity of a molecular chaperone. Molecular chaperones are a multifaceted class of proteins essential to protein homeostasis, and one of their major functions is to combat protein misfolding and aggregation, a phenomenon linked to a number of human disorders. In this work, we conjugated a small-molecule inhibitor of the aggregation of α-synuclein, a process associated with Parkinson’s disease (PD), to a specific cysteine residue on human Hsp70, a molecular chaperone with five free cysteines. We show that this regioselective conjugation augments in vitro the anti-aggregation activity of Hsp70 in a synergistic manner. This Hsp70 variant also displays in vivo an enhanced suppression of α-synuclein aggregation and its associated toxicity in a Caenorhabditis elegans model of PD.

Dual-Reactivity trans-Cyclooctenol Probes for Sulfenylation in Live Cells Enable Temporal Control via Bioorthogonal Quenching

Samuel L. Scinto, Oshini Ekanayake, Uthpala Seneviratne, Jessica E. Pigga, Samantha J. Boyd, Michael T. Taylor, Jun Liu, Christopher W. am Ende, Sharon Rozovsky, and Joseph M. Fox

Journal of the American Chemical Society, 2019 141 (28), 10932-10937
DOI: 10.1021/jacs.9b01164

Sulfenylation (RSH → RSOH) is a post-translational protein modification associated with cellular mechanisms for signal transduction and the regulation of reactive oxygen species. Protein sulfenic acids are challenging to identify and study due to their electrophilic and transient nature. Described here are sulfenic acid modifying trans-cycloocten-5-ol (SAM-TCO) probes for labeling sulfenic acid functionality in live cells. These probes enable a new mode of capturing sulfenic acids via transannular thioetherification, whereas “ordinary” trans-cyclooctenes react only slowly with sulfenic acids. SAM-TCOs combine with sulfenic acid forms of a model peptide and proteins to form stable adducts. Analogously, SAM-TCO with the selenenic acid form of a model protein leads to a selenoetherification product. Control experiments illustrate the need for the transannulation process coupled with the activated trans-cycloalkene functionality. Bioorthogonal quenching of excess unreacted SAM-TCOs with tetrazines in live cells provides both temporal control and a means of preventing artifacts caused by cellular-lysis. A SAM-TCO biotin conjugate was used to label protein sulfenic acids in live cells, and subsequent quenching by tetrazine prevented further labeling even under harshly oxidizing conditions. A cell-based proteomic study validates the ability of SAM-TCO probes to identify and quantify known sulfenic acid redox proteins as well as targets not captured by dimedone-based probes.

Rotational Freedom, Steric Hindrance, and Protein Dynamics Explain BLU554 Selectivity for the Hinge Cysteine of FGFR4

Lin, Xiaojing, Yosaatmadja, Yuliana, Kalyukina, Maria, Middleditch, Martin J., Zhang, Zhen, Lu, Xiaoyun, Ding, Ke, Patterson, Adam V., Smaill, Jeff B., Squire, Christopher J.

ACS Med. Chem. Lett., 2019
doi: 10.1021/acsmedchemlett.9b00196

Aberration in FGFR4 signaling drives carcinogenesis and progression in a subset of hepatocellular carcinoma (HCC) patients, thereby making FGFR4 an attractive molecular target for this disease. Selective FGFR4 inhibition can be achieved through covalently targeting a poorly conserved cysteine residue in the FGFR4 kinase domain. We report mass spectrometry assays and cocrystal structures of FGFR4 in covalent complex with the clinical candidate BLU554 and with a series of four structurally related inhibitors that define the inherent reactivity and selectivity profile of these molecules. We further reveal the structure of FGFR1 with one of our inhibitors and show that off-target covalent binding can occur through an alternative conformation that supports targeting of a cysteine conserved in all members of the FGFR family. Collectively, we propose that rotational freedom, steric hindrance, and protein dynamics explain the exceptional selectivity profile of BLU554 for targeting FGFR4.

Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition [@RobertoZoncu @DanNomura @OlzmannLab @Clive_chung @HijaiShin]

Clive Yik-Sham Chung, Hijai R. Shin, Charles A. Berdan, Breanna Ford, Carl C. Ward, James A. Olzmann, Roberto Zoncu & Daniel K. Nomura

Nature Chemical Biology, 2019 

doi: 10.1038/s41589-019-0308-4

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.