Covalent Inhibitors Research Articles

Discovery of Potent Covalent CRM1 Inhibitors Via a Customized Structure-Based Virtual Screening Pipeline and Bioassays.

CRM1 (chromosomal region maintenance 1, also referred to as exportin 1 or XPO1) plays a crucial role in maintaining the appropriate nuclear levels of tumor suppressor proteins (TSPs), growth regulatory proteins (GRPs), and antiapoptotic proteins, thereby contributing significantly to their anticancer effects. Dysregulation of CRM1-mediated nuclear transport, observed in a range of cancers such as colon cancer as well as autoimmune diseases, highlights its significance in various disease processes. In this paper, we employed a customized structure-based virtual screening campaign to search for novel covalent CRM1 inhibitors and purchased 50 potentially active compounds for in vitro bioassays. Among these candidates, AN-988 displayed a notably higher binding affinity (KD = 615 nM) toward CRM1, as determined by the biolayer interferometry (BLI) assay. Furthermore, AN-988 exhibited a strong suppression of colorectal cancer cell proliferation and remarkable anti-inflammatory effects. Notably, AN-988 induced cell apoptosis and cell cycle arrest in a time- and dose-dependent manner by effectively inhibiting the translocation of FOXO3a from the nucleus to the cytosol, thereby preserving the activity of FOXO3a. Collectively, our study identified AN-988 as a promising CRM1 inhibitor, underscoring its potential as a preclinical colon cancer therapy candidate.

CDK14 is regulated by IGF2BP2 and involved in osteogenic differentiation via Wnt/β-catenin signaling pathway in vitro

AimsCyclin-dependent kinase (CDK) family proteins involve in various cellular processes via regulating the cell cycle; however, their expression during osteogenic differentiation and postmenopausal osteoporosis remains poorly understood. Main methodsUsing bioinformatics, we screened for CDK14 bound to Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) and explored its expression in vitro with time-gradient model and in a mouse model of postmenopausal osteoporosis, building on prior research. Subsequently, we investigated its effect on osteoblast proliferation, cell cycle dynamics, and osteogenic differentiation by administering CDK14 siRNA and the covalent inhibitor FMF-04-159-2. Furthermore, we examined the interaction between IGF2BP2 and CDK14. Finally, we validated the regulatory role of CDK14 on the Wnt/β-catenin pathway. Key findingsOur findings demonstrate a time-dependent CDK14 expression patterns during osteogenic differentiation of MC3T3-E1 cell line, with an initial increase followed by gradual decline over time. Notably, CDK14 expression exhibited significant reduction in bone tissue of postmenopausal osteoporosis mouse model. CDK14 inhibition altered osteoblast cell cycle dynamics, significantly reduced cellular proliferation capacity, and impaired osteogenic differentiation ability. IGF2BP2 interacted with CDK14 mRNA, and stabilizing mRNA's structure and inhibiting its degradation. Additionally, CDK14 facilitated Low-density lipoprotein receptor-related protein 6 (LRP6) and Glycogen synthase kinase 3β (GSK3β) phosphorylation, thus regulating β-catenin levels. SignificanceThese findings provide further insight into the molecular mechanisms governing osteoblast proliferation, differentiation and osteoporosis.

Current Approaches and Novel Agents in the Treatment of Chronic Lymphocytic Leukemia.

The treatment of CLL has evolved from traditional chemoimmunotherapy (CIT) to an increasing number of targeted and biologic approaches. Randomized trials have demonstrated superiority of covalent bruton tyrosine kinase inhibitors (cBTKis) over CIT, and second-generation compounds such as acalabrutinib and zanubrutinib appear to have a more favorable efficacy/safety profile than ibrutinib. The noncovalent BTKi, pirtobrutinib, has shown impressive activity after failure of the cBTKis and is quite tolerable. The Bcl-2 inhibitor venetoclax plus a CD20, generally obinutuzumab, provides a high level of efficacy as initial treatment or after failure on a cBTKi, with many patients achieving a state of undetectable minimal residual disease. Promising novel approaches include BTK degraders, bispecific antibodies, and chimeric antigen receptor T-cell (CAR-T)-cell therapy. What is clear is that CIT is archaic, and current and future targeted approaches will continue to improve the outcome for patients with chronic lymphocytic leukemia.

Adagrasib in KRYSTAL-12has Broken the KRAS G12C Enigma Code in Non-Small Cell Lung Carcinoma.

Kirsten rat sarcoma viral oncogene homolog (KRAS) G12C-mutant non-small cell lung carcinoma (NSCLC) accounts for approximately 10-13% of advanced nonsquamous NSCLC cases in Western populations, presenting a significant therapeutic challenge owing to the difficulty of directly targeting KRAS. Adagrasib, an oral small-molecule covalent inhibitor, irreversibly and selectively targets KRASG12C in its inactive state. It received accelerated Food and Drug Administration (FDA) approval on December 12, 2022, following the KRYSTAL-1 Phase II trial. The Phase III KRYSTAL-12 trial demonstrated that adagrasib significantly improved median progression-free survival (mPFS) compared with docetaxel (HR, 0.58; 95% CI: 0.45-0.76; P<0.0001) and increased the intracranial objective response rate (ORR) to 40% in the central nervous system (CNS) evaluable population. This paper evaluates the clinical efficacy of adagrasib in KRAS G12C-mutated advanced NSCLC discussing its potential advantages over other inhibitors such as sotorasib. Despite not reaching the 6-month mPFS benchmark, adagrasib offers significant clinical benefits, particularly for the management of CNS metastases. In this pros and cons debate, we argue that adagrasib has broken the KRAS G12C enigma code in NSCLC.

401 (PB389): Discovery of a covalent inhibitor targeting the undruggable oncogene cMyc

401 (PB389): Discovery of a covalent inhibitor targeting the undruggable oncogene cMyc

Design and development of a series of 4-(piperazin-1-yl)pyrimidines as irreversible menin inhibitors

The interaction between menin and MLL1 protein plays an important role in AML with MLL rearrangement and NPM1 mutation. Blocking the formation of menin-MLL complex can inhibit proliferation and induce differentiation in these cancer subtypes. In development of anticancer drugs, irreversible inhibitors are gaining spotlight as they may have better activities than the reversible analogs. Therefore, we designed and developed a novel series of covalent menin inhibitors. Among these compounds, 37 emerges as a selective and potent inhibitor of MLL fusion protein-expressing leukemic cells. The cellular study indicates 37 has a distinct mechanism of action, in both reducing menin protein levels and downregulating MEN1 transcription. This effect of 37 is not involved in proteasomal degradation, and may directly affect the synthesis of menin protein, which offers a significant advantage in addressing acquired resistance to menin inhibitors. Further study showed that compound 37 has prolonged anti-leukemic action and exhibits promising in vivo efficacy, making it a valuable probe for further menin-MLL interaction studies.

Prominent Neuroprotective Potential of Indole-2-N-methylpropargylamine: High Affinity and Irreversible Inhibition Efficiency towards Monoamine Oxidase B Revealed by Computational Scaffold Analysis

Background: Monoamine oxidases (MAO) are flavoenzymes that metabolize a range of brain neurotransmitters, whose dysregulation is closely associated with the development of various neurological disorders. This is why MAOs have been the central target in pharmacological interventions for neurodegeneration for more than 60 years. Still, existing drugs only address symptoms and not the cause of the disease, which underlines the need to develop more efficient inhibitors without adverse effects. Methods: Our drug design strategy relied on docking 25 organic scaffolds to MAO-B, which were extracted from the ChEMBL20 database with the highest cumulative counts of unique member compounds and bioactivity assays. The most promising candidates were substituted with the inactivating propargylamine group, while further affinity adjustment was made by its N-methylation. A total of 46 propargylamines were submitted to the docking and molecular dynamics simulations, while the best binders underwent mechanistic DFT analysis that confirmed the hydride abstraction mechanism of the covalent inhibition reaction. Results: We identified indole-2-propargylamine 4fH and indole-2-N-methylpropargylamine 4fMe as superior MAO-B binders over the clinical drugs rasagiline and selegiline. DFT calculations highlighted 4fMe as more potent over selegiline, evident in a reduced kinetic requirement (ΔΔG‡ = −2.5 kcal mol−1) and an improved reaction exergonicity (ΔΔGR = −4.3 kcal mol−1), together with its higher binding affinity, consistently determined by docking (ΔΔGBIND = −0.1 kcal mol−1) and MM-PBSA analysis (ΔΔGBIND = −1.5 kcal mol−1). Conclusions: Our findings strongly advocate 4fMe as an excellent drug candidate, whose synthesis and biological evaluation are highly recommended. Also, our results reveal the structural determinants that influenced the affinity and inhibition rates that should cooperate when designing further MAO inhibitors, which are of utmost significance and urgency with the increasing prevalence of brain diseases.

Identification of Potent, Broad-Spectrum Coronavirus Main Protease Inhibitors for Pandemic Preparedness.

The COVID-19 pandemic highlights the ongoing risk of zoonotic transmission of coronaviruses to global health. To prepare for future pandemics, it is essential to develop effective antivirals targeting a broad range of coronaviruses. Targeting the essential and clinically validated coronavirus main protease (Mpro), we constructed a structurally diverse Mpro panel by clustering all known coronavirus sequences by Mpro active site sequence similarity. Through screening, we identified a potent covalent inhibitor that engaged the catalytic cysteine of SARS-CoV-2 Mpro and used structure-based medicinal chemistry to develop compounds in the pyrazolopyrimidine sulfone series that exhibit submicromolar activity against multiple Mpro homologues. Additionally, we solved the first X-ray cocrystal structure of Mpro from the human-infecting OC43 coronavirus, providing insights into potency differences among compound-target pairs. Overall, the chemical compounds described in this study serve as starting points for the development of antivirals with broad-spectrum activity, enhancing our preparedness for emerging human-infecting coronaviruses.

Discovery of electrophilic degraders that exploit SNAr chemistry.

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly 'undruggable' targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

Development of Novel Indole-Based Covalent Inhibitors of TEAD as Potential Antiliver Cancer Agents.

Abnormal activation of the YAP transcriptional signaling pathway drives proliferation in many hepatocellular carcinoma (HCC) and hepatoblastoma (HB) cases. Current treatment options often face resistance and toxicity, highlighting the need for alternative therapies. This article reports the discovery of a hit compound C-3 from docking-based virtual screening targeting TEAD lipid binding pocket, which inhibited TEAD-mediated transcription. Optimization led to the identification of a potent and covalent inhibitor CV-4-26 that exhibited great antitumor activity in HCC and HB cell lines in vitro, xenografted human HCC, and murine HB in vivo. These outcomes signify the potential of a highly promising therapeutic candidate for addressing a subset of HCC and HB cancers. In the cases of current treatment challenges due to high upregulation of YAP-TEAD activity, these findings offer a targeted alternative for more effective interventions against liver cancer.

Potent covalent irreversible inhibitor of KRAS G12C IBI351 in patients with advanced solid tumors: First-in-human phase I study

BackgroundIBI351 is an irreversible and covalent inhibitor of KRAS G12C. Despite FDA approval of two KRAS G12C inhibitors, there are still significant unmet clinical needs in Chinese patients and ongoing concerns about the optimal dosage. Herein, we presented the phase Ia/Ib study of IBI351 monotherapy in Chinese patients with advanced solid tumors harboring KRAS G12C mutation. MethodsIn phase Ia dose escalation, IBI351 at 250/450/700/900 mg once daily and 450/600/750 mg twice daily (BID) were evaluated. Potentially efficacious doses and optimal recommended phase 2 dose (RP2D) were further evaluated in patients with advanced non-small cell lung cancer (NSCLC) in phase Ia dose expansion and phase Ib. Safety, pharmacokinetics, and investigator-assessed tumor response were evaluated. ResultsAs of June 13, 2023, 176 patients were enrolled. IBI351 was well tolerated with no dose-limiting toxicity reported across all evaluated doses. The RP2D was determined as 600 mg BID by considering safety, efficacy and pharmacokinetics. A total of 168 patients (95.5 %) had at least one treatment-related adverse event (TRAE), and 64 patients (36.4 %) had grade 3 or higher TRAEs, most commonly gamma-glutamyl transferase increased (10.2 %) and anemia (6.8 %). For patients with NSCLC, the confirmed objective response rate (ORR) was 45.5 % across all doses. At 600 mg BID, the confirmed ORR was 46.8 % and median progression-free survival was 9.6 months with a median follow-up of 6.9 months. ConclusionsIBI351 was well tolerated in patients with advanced solid tumors and showed promising antitumor activity in advanced NSCLC patients with KRAS G12C mutation.

Insights into the Main Protease of SARS-CoV-2: Thermodynamic Analysis, Structural Characterization, and the Impact of Inhibitors.

The main protease (Mpro) of SARS-CoV-2 is an essential enzyme for coronaviral maturation and is the target of Paxlovid, which is currently the standard-of-care treatment for COVID-19. There remains a need to identify new inhibitors of Mpro as viral resistance to Paxlovid emerges. Here, we report the use of native mass spectrometry coupled with 193 nm ultraviolet photodissociation (UVPD) and integrated with other biophysical tools to structurally characterize Mpro and its interactions with potential covalent inhibitors. The overall energy landscape was obtained using variable temperature nanoelectrospray ionization (vT-nESI), thus providing quantitative evaluation of inhibitor binding on the stability of Mpro. Thermodynamic parameters extracted from van't Hoff plots revealed that the dimeric complexes containing each inhibitor showed enhanced stability through increased melting temperatures as well as overall lower average charge states, giving insight into the basis for inhibition mechanisms.

Advances in the Search for SARS-CoV-2 Mpro and PLpro Inhibitors.

SARS-CoV-2 is a spherical, positive-sense, single-stranded RNA virus with a large genome, responsible for encoding both structural proteins, vital for the viral particle's architecture, and non-structural proteins, critical for the virus's replication cycle. Among the non-structural proteins, two cysteine proteases emerge as promising molecular targets for the design of new antiviral compounds. The main protease (Mpro) is a homodimeric enzyme that plays a pivotal role in the formation of the viral replication-transcription complex, associated with the papain-like protease (PLpro), a cysteine protease that modulates host immune signaling by reversing post-translational modifications of ubiquitin and interferon-stimulated gene 15 (ISG15) in host cells. Due to the importance of these molecular targets for the design and development of novel anti-SARS-CoV-2 drugs, the purpose of this review is to address aspects related to the structure, mechanism of action and strategies for the design of inhibitors capable of targeting the Mpro and PLpro. Examples of covalent and non-covalent inhibitors that are currently being evaluated in preclinical and clinical studies or already approved for therapy will be also discussed to show the advances in medicinal chemistry in the search for new molecules to treat COVID-19.

Targeting cancer with small-molecule pan-KRAS degraders

Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.

A covalent creatine kinase inhibitor ablates glioblastoma migration and sensitizes tumors to oxidative stress

Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

Elucidating the resistance mechanisms and binding pattern of novel Oxa-48-like carbapenemases covalent inhibitors: A hybrid experimental and in silico approach

Klebsiella pneumoniae is a Gram negative bacteria that can cause harm to the public's health and vulnerability has increased due to the emergence of highly virulent strains resistant to carbapenem antibiotics. The K. pneumoniae resistance mechanism involves extended-spectrum beta-lactamases, which are enzymes that can break down a broad range of antibiotics. The particular concern is Class D β-lactamases like OXA-48, which further complicates treatment options for infections caused by K. pneumoniae. The current study screened 7800 samples from three hospitals in Pakistan and found 353 positive samples of K. pneumoniae. The isolates were resistant to SXT, AMP, FEP CTX, CIP, TET, MEM, and IPM, while effective antibiotics were TGC, FOS, FOX, SCF and TZP. The sequencing and mutational analysis of the OXA-48 gene revealed 45-point mutations, including five novel missense mutations (Thr104:Ala104, Asn110:Asp110, Glu168:Gln168, Ser171:Ala171 and Arg214:Ser214). These mutations can contribute to the antibiotic affinity decrease and cause resistance. Furthermore, the study aimed to develop covalent inhibitors of OXA-48 (wild type and mutant) carbapenemases using a combination of molecular docking and molecular dynamic (MD) simulation approaches from natural products from the ZNINC20 database. 10 compounds (ZINC103533306, ZINC103531564, ZINC3861001, ZINC103533375, ZINC12411532, ZINC103533290, ZINC12376465, ZINC85875819, ZINC59586513 and ZINC13370550) showed best fit and high interactions with the OXA-48 active pocket. The binding free energies calculated from the MD showed that these compounds have high affinity with both the wild type and mutated OXA-48 protein. The selected compounds showed good drug-like properties and can be the leading inhibitors to combat the resistant K. pneumoniae strains.

Synthesis, antimicrobial, anticancer activity, 3D QSAR, ADMET properties, and in silico target fishing of novel N,N-disubstituted chloroacetamides

The recent FDA approval of afatanib, ibrutinib, and osimertinib, which covalently bind to specific cysteine residues in target kinases, has renewed interest in covalent drug discovery. Besides α,β-unsaturated carbonyls, chloroacetamides have emerged as popular warheads for designing targeted covalent inhibitors. In this study, we synthesized thirteen N,N-disubstituted chloroacetamides (1–13) by acylating secondary amines with chloroacetyl chloride, selecting substituents to provide a wide range of lipophilicity. We evaluated their anticancer and antimicrobial activity, finding five compounds with significant cytotoxicity against HeLa, K562, and A549 cell lines (IC50 <10 μM). Notably, compound 10 activated caspases 3, 8, and 9, promoting both intrinsic and extrinsic apoptotic pathways, while compounds 9–12 were strong apoptosis inducers. A 3D QSAR model showed that aromatic substituents on nitrogen atoms reduced HeLa potency, whereas the overall molecular shape had a positive effect. ChemBL and pharmacophoric similarity searches suggested potential anticancer targets, including alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), glycogen synthase kinase-3β, and calmodulin. Docking studies indicated that chloroacetamides bind to ADH and ALDH via hydrogen bonds and hydrophobic interactions. Pharmacokinetics predictions suggest that chloroacetamides are druglike molecules with promising ADMET properties.

Structure-Based Optimization of a Series of Covalent, Cell Active Bfl-1 Inhibitors.

Bfl-1, a member of the Bcl-2 family of proteins, plays a crucial role in apoptosis regulation and has been implicated in cancer cell survival and resistance to venetoclax therapy. Due to the unique cysteine residue in the BH3 binding site, the development of covalent inhibitors targeting Bfl-1 represents a promising strategy for cancer treatment. Herein, the optimization of a covalent cellular tool from a lead-like hit using structure based design is described. Informed by a reversible X-ray fragment screen, the strategy to establish interactions with a key glutamic acid residue (Glu78) and optimize binding in a cryptic pocket led to a 1000-fold improvement in biochemical potency without increasing reactivity of the warhead. Compound (R,R,S)-26 has a kinact/KI of 4600 M-1 s-1, shows <1 μM caspase activation in a cellular assay and cellular target engagement, and has good physicochemical properties and a promising in vivo profile.

Advances in reversible covalent kinase inhibitors.

Reversible covalent kinase inhibitors (RCKIs) are a class of novel kinase inhibitors attracting increasing attention because they simultaneously show the selectivity of covalent kinase inhibitors yet avoid permanent protein-modification-induced adverse effects. Over the last decade, RCKIs have been reported to target different kinases, including Atypical group of kinases. Currently, three RCKIs are undergoing clinical trials. Here, advances in RCKIs are reviewed to systematically summarize the characteristics of electrophilic groups, chemical scaffolds, nucleophilic residues, and binding modes. In so doing, we integrate key insights into privileged electrophiles, the distribution of nucleophiles, and hence effective design strategies for the development of RCKIs. Finally, we provide a further perspective on future design strategies for RCKIs, including those that target proteins other than kinases.

Beyond KRAS(G12C): Biochemical and Computational Characterization of Sotorasib and Adagrasib Binding Specificity and the Critical Role of H95 and Y96.

Mutated KRAS proteins are frequently expressed in some of the most lethal human cancers and thus have been a target of intensive drug discovery efforts for decades. Lately, KRAS(G12C) switch-II pocket (SII-P)-targeting covalent small molecule inhibitors have finally reached clinical practice. Sotorasib (AMG-510) was the first FDA-approved covalent inhibitor to treat KRAS(G12C)-positive nonsmall cell lung cancer (NSCLC), followed soon by adagrasib (MRTX849). Both drugs target the GDP-bound state of KRAS(G12C), exploiting the strong nucleophilicity of acquired cysteine. Here, we evaluate the similarities and differences between sotorasib and adagrasib in their RAS SII-P binding by applying biochemical, cellular, and computational methods. Exact knowledge of SII-P engagement can enable targeting this site by reversible inhibitors for KRAS mutants beyond G12C. We show that adagrasib is strictly KRAS- but not KRAS(G12C)-specific due to its strong and unreplaceable interaction with H95. Unlike adagrasib, sotorasib is less dependent on H95 for its binding, making it a RAS isoform-agnostic compound, having a similar functionality also with NRAS and HRAS G12C mutants. Our results emphasize the accessibility of SII-P beyond oncogenic G12C and aid in understanding the molecular mechanism behind the clinically observed drug resistance, associated especially with secondary mutations on KRAS H95 and Y96.