A Warhead Substitution Study on the Coronavirus Main Protease Inhibitor Nirmatrelvir.

The SARS-CoV-2 pandemic is currently causing an unprecedented global health emergency since its emergence in December 2019. In December 2021, the FDA granted emergency use authorization to nirmatrelvir, a SARS-CoV-2 main protease inhibitor, for treating infected patients. This peptidomimetic is designed with a nitrile warhead, which forms a covalent bond to the viral protease. Herein, we investigate nirmatrelvir analogs with different warheads and their inhibitory activities. In addition, antiviral activities against human alphacoronavirus 229E was also investigated along with a cell-based assay. We discovered that the hydroxymethylketone and ketobenzothiazole warheads were equipotent to the nitrile warhead, suggesting that these analogs can also be used for treating coronavirus infections.

Structure-activity relationship studies of allosteric inhibitors of EYA2 tyrosine phosphatase.

Human eyes absent (EYA) proteins possess Tyr phosphatase activity, which is critical for numerous cancer and metastasis promoting activities, making it an attractive target for cancer therapy. In this work, we demonstrate that the inhibitor-bound form of EYA2 does not favour binding to Mg2+ , which is indispensable for the Tyr phosphatase activity. We further describe characterization and optimization of this class of allosteric inhibitors. A series of analogues were synthesized to improve potency of the inhibitors and to elucidate structure-activity relationships. Two co-crystal structures confirm the binding modes of this class of inhibitors. Our medicinal chemical, structural, biochemical, and biophysical studies provide insight into the molecular interactions of EYA2 with these allosteric inhibitors. The compounds derived from this study are useful for exploring the function of the Tyr phosphatase activity of EYA2 in normal and cancerous cells and serve as reference compounds for screening or developing allosteric phosphatase inhibitors. Finally, the co-crystal structures reported in this study will aid in structure-based drug discovery against EYA2.

Fragment-based lead discovery of indazole-based compounds as AXL kinase inhibitors.

AXL is a member of the TAM (TYRO3, AXL, MER) subfamily of receptor tyrosine kinases. It is upregulated in a variety of cancers and its overexpression is associated with poor disease prognosis and acquired drug resistance. Utilizing a fragment-based lead discovery approach, a new indazole-based AXL inhibitor was obtained. The indazole fragment hit 11, identified through a high concentration biochemical screen, was expeditiously improved to fragment 24 by screening our in-house expanded library of fragments (ELF) collection. Subsequent fragment optimization guided by docking studies provided potent inhibitor 54 with moderate exposure levels in mice. X-ray crystal structure of analog 50 complexed with the I650M mutated kinase domain of Mer revealed the key binding interactions for the scaffold. The good potency coupled with reasonable kinase selectivity, moderate in vivo exposure levels, and availability of structural information for the series makes it a suitable starting point for further optimization efforts.

A head-to-head comparison of the inhibitory activities of 15 peptidomimetic SARS-CoV-2 3CLpro inhibitors.

The COVID-19 pandemic caused by SARS-CoV-2 has created an unprecedented global health emergency. As of July 2021, only three antiviral therapies have been approved by the FDA for treating infected patients, highlighting the urgent need for more antiviral drugs. The SARS-CoV-2 3CL protease (3CLpro) is deemed an attractive drug target due to its essential role in viral polyprotein processing and pathogenesis. Indeed, a number of peptidomimetic 3CLpro inhibitors armed with electrophilic warheads have been reported by various research groups that can potentially be developed for treating COVID-19. However, it is currently impossible to compare their relative potencies due to the different assays employed. To solve this, we conducted a head-to-head comparison of fifteen reported peptidomimetic inhibitors in a standard FRET-based SARS-CoV-2 3CLpro inhibition assay to compare and identify potent inhibitors for development. Inhibitor design and the suitability of various warheads are also discussed.

Strategic Design of Catalytic Lysine-Targeting Reversible Covalent BCR-ABL Inhibitors*.

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid (CBA) warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved potency against both wild-type and mutant ABL kinases, especially ABLT315I bearing the gatekeeper residue mutation. We show the evolutionarily conserved lysine can be targeted selectively, and the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate off-targets of our compounds at proteome-wide level in different mammalian cells.

Stepwise Evolution of Fragment Hits against MAPK Interacting Kinases 1 and 2.

Dysregulation of translation initiation factor 4E (eIF4E) activity occurs in various cancers. Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) play a fundamental role in activation of eIF4E. Structure-activity relationship-driven expansion of a fragment hit led to discovery of dual MNK1 and MNK2 inhibitors based on a novel pyridine-benzamide scaffold. The compounds possess promising in vitro and in vivo pharmacokinetic profiles and show potent on target inhibition of eIF4E phosphorylation in cells.

Correction to "Discovery of Irreversible Inhibitors Targeting Histone Methyltransferase, SMYD3".

[This corrects the article DOI: 10.1021/acsmedchemlett.9b00170.].

Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg2+ binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.

Discovery of Irreversible Inhibitors Targeting Histone Methyltransferase, SMYD3.

SMYD3 is a histone methyltransferase that regulates gene transcription, and its overexpression is associated with multiple human cancers. A novel class of tetrahydroacridine compounds which inhibit SMYD3 through a covalent mechanism of action is identified. Optimization of these irreversible inhibitors resulted in the discovery of 4-chloroquinolines, a new class of covalent warheads. Tool compound 29 exhibits high potency by inhibiting SMYD3's enzymatic activity and showing antiproliferative activity against HepG2 in 3D cell culture. Our findings suggest that covalent inhibition of SMYD3 may have an impact on SMYD3 biology by affecting expression levels, and this warrants further exploration.

Targeting cancer addiction for SALL4 by shifting its transcriptome with a pharmacologic peptide.

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable.

Optimization of Selective Mitogen-Activated Protein Kinase Interacting Kinases 1 and 2 Inhibitors for the Treatment of Blast Crisis Leukemia.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by bcr-abl1, a constitutively active tyrosine kinase fusion gene responsible for an abnormal proliferation of leukemic stem cells (LSCs). Inhibition of BCR-ABL1 kinase activity offers long-term relief to CML patients. However, for a proportion of them, BCR-ABL1 inhibition will become ineffective at treating the disease, and CML will progress to blast crisis (BC) CML with poor prognosis. BC-CML is often associated with excessive phosphorylated eukaryotic translation initiation factor 4E (eIF4E), which renders LSCs capable of proliferating via self-renewal, oblivious to BCR-ABL1 inhibition. In vivo, eIF4E is exclusively phosphorylated on Ser209 by MNK1/2. Consequently, a selective inhibitor of MNK1/2 should reduce the level of phosphorylated eIF4E and re-sensitize LSCs to BCR-ABL1 inhibition, thus hindering the proliferation of BC LSCs. We report herein the structure-activity relationships and pharmacokinetic properties of a selective MNK1/2 inhibitor clinical candidate, ETC-206, which in combination with dasatinib prevents BC-CML LSC self-renewal in vitro and enhances dasatinib antitumor activity in vivo.

Backbone resonance assignments for the SET domain of human methyltransferase NSD3 in complex with its cofactor.

NSD3 is a histone H3 methyltransferase that plays an important role in chromatin biology. A construct containing the methyltransferase domain encompassing residues Q1049-K1299 of human NSD3 was obtained and biochemical activity was demonstrated using histone as a substrate. Here we report the backbone HN, N, Cα, C', and side chain Cß assignments of the construct in complex with S-adenosyl-L-methionine (SAM). Based on these assignments, secondary structures of NSD3/SAM complex in solution were determined.

Discovery and characterisation of the automethylation properties of PRDM9.

We have previously characterised the histone lysine methyltransferase properties of PRDM9, a member of the PRDM family of putative transcriptional regulators. PRDM9 displays broad substrate recognition and methylates a range of histone substrates, including octamers, core histone proteins, and peptides. In the present study, we show that PRDM9 performs intramolecular automethylation on multiple lysine residues localised to a lysine-rich region on the post-SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain. PRDM9 automethylation is abolished by a single active-site mutation, C321P, also known to disrupt interactions with S-adenosylmethionine. We have taken an initial step towards tool compound generation through rational design of a substrate-mimic, peptidic inhibitor of PRDM9 automethylation. The discovery of automethylation in PRDM9 adds a new dimension to our understanding of PRDM9 enzymology.

Miniature bovine pancreatic trypsin inhibitors (m-BPTIs) of the West Nile virus NS2B-NS3 protease.

The mosquito-borne West Nile virus (WNV) causes a wide range of symptoms ranging from fever to the often fatal viral encephalitis. To date, no vaccine or drug therapy is available. The trypsin-like WNV NS2B-NS3 protease is deemed a plausible drug target and was shown to be inhibited by bovine pancreatic trypsin inhibitor (BPTI), a 58-residue protein isolated from bovine lung. Herein, we report a protein truncation study that resulted in a novel 14-residue cyclic peptide with equipotent inhibitory activity to native BPTI. We believe our truncation strategy can be further applied in the development of peptide-based inhibitors targeting trypsin-like proteases.

Structure-Activity Relationship Studies of Mitogen Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 and BCR-ABL1 Inhibitors Targeting Chronic Myeloid Leukemic Cells.

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.

Identification of covalent active site inhibitors of dengue virus protease.

Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.

Antiviral activities of 15 dengue NS2B-NS3 protease inhibitors using a human cell-based viral quantification assay.

The dengue virus is a mosquito-borne pathogen responsible for an estimated 50-100 million human dengue infections annually. There are currently no approved drugs against this disease, resulting in a major unmet clinical need. The dengue viral NS2B-NS3 protease has been identified as a plausible drug target due to its involvement in viral replication in mammalian host cells. In the past decade, at least 20 dengue NS2B-NS3 protease inhibitors have been reported in the literature with a range of inhibitory activities in protease assays. However, such assays do not shed light on an inhibitor's ability to penetrate human cell membranes where the viral protease resides. In this study, we investigated the antiviral activities of 15 small-molecule and peptide-based NS2B-NS3 inhibitors on dengue serotype 2-infected HuH-7 human hepatocarcinoma cells. Experimental results revealed anthraquinone ARDP0006 (compound 5) to be the most potent inhibitor which reduced dengue viral titer by more than 1 log PFU/mL at 1 µM in our cell-based assays involving HuH-7 and K562 cell lines, suggesting that its scaffold could serve as a lead for further medicinal chemistry studies. Compound 5 was also found to be non-cytotoxic at 1 µM over 3 days incubation on HuH-7 cells using the Alamar Blue cellular toxicity assay.

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques.

PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.

A P2 and P3 substrate specificity comparison between the Murray Valley encephalitis and West Nile virus NS2B/NS3 protease using C-terminal agmatine dipeptides.

The Murray Valley encephalitis virus (MVEV) and the West Nile virus (WNV) are mosquito-borne single-stranded RNA Flaviviruses responsible for many cases of viral encephalitis and deaths worldwide. The former is endemic in north Australia and Papua New Guinea while the latter has spread to different parts of the world and was responsible for a recent North American outbreak in 2012, resulting in 243 fatalities. There is currently no approved vaccines or drugs against MVEV and WNV viral infections. A plausible drug target is the viral non-structural NS2B/NS3 protease due to its role in viral replication. This trypsin-like serine protease recognizes and cleaves viral polyproteins at the C-terminal end of an arginine residue, opening an avenue for the development of peptide-based antivirals. This communication compares the P2 and P3 residue preferences of the MVEV and WNV NS2B/NS3 proteases using a series of C-terminal agmatine dipeptides. Our results revealed that both viral enzymes were highly specific toward lysines at the P2 and P3 positions, suggesting that a peptidomimetic viral protease inhibitor developed against one virus should also be active against the other.

Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV.