Covalent fragment libraries in drug discovery-Design, synthesis, and screening methods.

As the development of drugs with a covalent mode of action is becoming increasingly popular, well-validated covalent fragment-based drug discovery (FBDD) methods have been comparatively slow to keep up with the demand. In this chapter the principles of covalent fragment reactivity, library design, synthesis, and screening methods are explored in depth, focussing on literature examples with direct applications to practical covalent fragment library design and screening. Further, questions about the future of the field are explored and potential useful advances are proposed.

Antibacterial Activity of Ebselen.

Ebselen is a low-molecular-weight organoselenium compound that has been broadly studied for its antioxidant, anti-inflammatory, and cytoprotective properties. These advantageous properties were initially associated with mimicking the activity of selenoprotein glutathione peroxidase, but the biomedical impact of this compound appear to be far more complex. Ebselen serves as a substrate or inhibitor with multiple protein/enzyme targets, whereas inhibition typically originates from the covalent modification of cysteine residues by opening the benzisoselenazolone ring and S-Se bond formation. The inhibition of enzymes of various classes and origins has been associated with substantial antimicrobial potential among other activities. In this contribution, we summarize the current state of the art regarding the antibacterial activity of ebselen. This activity, alone and in combination with commercial pharmaceuticals, against pathogens, including those resistant to drugs, is presented, together with the molecular mechanism behind the reactivity. The specific inactivation of thioredoxin reductase, bacterial toxins, and other resistance factors is considered to have certain therapeutic implications. Synergistic action and sensitization to common antibiotics assisted with the use of ebselen appear to be promising directions in the treatment of persistent infections.

Insights into the Inhibitory Mechanisms of the Covalent Drugs for DNMT3A.

The perturbations of DNA methyltransferase 3 alpha (DNMT3A) may cause uncontrolled gene expression, resulting in cancers and tumors. The DNMT inhibitors Azacytidine (AZA) and Zebularine (ZEB) inhibit the DNMT family with no specificities, and consequently would bring side effects during the treatment. Therefore, it is vital to understand the inhibitory mechanisms in DNMT3A to inform the new inhibitor design for DNMTs. Herein, we carried out molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations to investigate the inhibitory mechanisms of the AZA and ZEB. The results were compared to the methyl transfer of cytosine. We showed how the AZA might stop the methyl transfer process, whereas the ZEB might be stuck in a methyl-transferred intermediate (IM3). The IM3 state then fails the elimination due to the unique protein dynamics that result in missing the catalytic water chain. Our results brought atomic-level insights into the mechanisms of the two drugs in DNMT3A, which could benefit the new generation of drug design for the DNMTs.

An update on the discovery and development of reversible covalent inhibitors.

Small molecule drugs that covalently bind irreversibly to their target proteins have several advantages over conventional reversible inhibitors. They include increased duration of action, less-frequent drug dosing, reduced pharmacokinetic sensitivity, and the potential to target intractable shallow binding sites. Despite these advantages, the key challenges of irreversible covalent drugs are their potential for off-target toxicities and immunogenicity risks. Incorporating reversibility into covalent drugs would lead to less off-target toxicity by forming reversible adducts with off-target proteins and thus reducing the risk of idiosyncratic toxicities caused by the permanent modification of proteins, which leads to higher levels of potential haptens. Herein, we systematically review electrophilic warheads employed during the development of reversible covalent drugs. We hope the structural insights of electrophilic warheads would provide helpful information to medicinal chemists and aid in designing covalent drugs with better on-target selectivity and improved safety.

Drug Repurposing against KRAS Mutant G12C: A Machine Learning, Molecular Docking, and Molecular Dynamics Study.

The Kirsten rat sarcoma viral G12C (KRASG12C) protein is one of the most common mutations in non-small-cell lung cancer (NSCLC). KRASG12C inhibitors are promising for NSCLC treatment, but their weaker activity in resistant tumors is their drawback. This study aims to identify new KRASG12C inhibitors from among the FDA-approved covalent drugs by taking advantage of artificial intelligence. The machine learning models were constructed using an extreme gradient boosting (XGBoost) algorithm. The models can predict KRASG12C inhibitors well, with an accuracy score of validation = 0.85 and Q2Ext = 0.76. From 67 FDA-covalent drugs, afatinib, dacomitinib, acalabrutinib, neratinib, zanubrutinib, dutasteride, and finasteride were predicted to be active inhibitors. Afatinib obtained the highest predictive log-inhibitory concentration at 50% (pIC50) value against KRASG12C protein close to the KRASG12C inhibitors. Only afatinib, neratinib, and zanubrutinib covalently bond at the active site like the KRASG12C inhibitors in the KRASG12C protein (PDB ID: 6OIM). Moreover, afatinib, neratinib, and zanubrutinib exhibited a distance deviation between the KRASG2C protein-ligand complex similar to the KRASG12C inhibitors. Therefore, afatinib, neratinib, and zanubrutinib could be used as drug candidates against the KRASG12C protein. This finding unfolds the benefit of artificial intelligence in drug repurposing against KRASG12C protein.

Efficacy of a Covalent Microtubule Stabilizer in Taxane-Resistant Ovarian Cancer Models.

Ovarian cancer often has a poor clinical prognosis because of late detection, frequently after metastatic progression, as well as acquired resistance to taxane-based therapy. Herein, we evaluate a novel class of covalent microtubule stabilizers, the C-22,23-epoxytaccalonolides, for their efficacy against taxane-resistant ovarian cancer models in vitro and in vivo. Taccalonolide AF, which covalently binds ß-tubulin through its C-22,23-epoxide moiety, demonstrates efficacy against taxane-resistant models and shows superior persistence in clonogenic assays after drug washout due to irreversible target engagement. In vivo, intraperitoneal administration of taccalonolide AF demonstrated efficacy against the taxane-resistant NCI/ADR-RES ovarian cancer model both as a flank xenograft, as well as in a disseminated orthotopic disease model representing localized metastasis. Taccalonolide-treated animals had a significant decrease in micrometastasis of NCI/ADR-RES cells to the spleen, as detected by quantitative RT-PCR, without any evidence of systemic toxicity. Together, these findings demonstrate that taccalonolide AF retains efficacy in taxane-resistant ovarian cancer models in vitro and in vivo and that its irreversible mechanism of microtubule stabilization has the unique potential for intraperitoneal treatment of locally disseminated taxane-resistant disease, which represents a significant unmet clinical need in the treatment of ovarian cancer patients.

Introducing of potent cytotoxic novel 2-(aroylamino)cinnamamide derivatives against colon cancer mediated by dual apoptotic signal activation and oxidative stress.

Curcumin and trans-cinnamaldehyde are acrolein-based Michael acceptor compounds that are commonly found in domestic condiments, and known to cause cancer cell death via redox mechanisms. Based on the structural features of these compounds we designed and synthesized several 2-cinnamamido-N-substituted-cinnamamide (bis-cinnamamide) compounds. One of the derivatives, (Z)-2-[(E)-cinnamamido]-3-phenyl-N-propylacrylamide 8 showed a moderate antiproliferative potency (HCT-116 cell line inhibition of 32.0 µM), no inhibition of normal cell lines C-166, and proven cellular activities leading to apoptosis. SAR studies led to more than 10-fold increase in activity. Our most promising compound, [(Z)-3-(1H-indol-3-yl)-N-propyl-2-[(E)-3-(thien-2-yl)propenamido)propenamide] 45 killed colon cancer cells at IC50 = 0.89 µM (Caco-2), 2.85 µM (HCT-116) and 1.65 µM (HT-29), while exhibiting much weaker potency on C-166 and BHK normal cell lines (IC50 = 71 µM and 77.6 µM, respectively). Cellular studies towards identifying the compounds mechanism of cytotoxic activities revealed that apoptotic induction occurs in part as a result of oxidative stress. Importantly, the compounds showed inhibition of cancer stem cells that are critical for maintaining the potential for self-renewal and stemness. The results presented here show discovery of covalently acting Michael addition compounds that potently kill cancer cells by a defined mechanism, with prominent selectivity profile over non-cancerous cell lines.

Development of covalent NLRP3 inflammasome inhibitors: Chemistry and biological activity.

The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is the best recognized and most widely implicated regulator of caspase-1 activation. It is a key regulator of innate immune response and is involved in many pathophysiological processes. Recent evidences for its inappropriate activation in autoinflammatory, autoimmune, as well as in neurodegenerative diseases attract a growing interest toward the development of small molecules NLRP3 inhibitors. Based on the knowledge of biochemical and structural aspects of NLRP3 activation, one successful strategy in the identification of NLRP3 inhibitors relies on the development of covalent irreversible inhibitors. Covalent inhibitors are reactive electrophilic molecules able to alkylate nucleophiles in the target protein. These inhibitors could ensure good efficacy and prolonged duration of action both in vitro and in vivo. In spite of these advantages, effects on other signalling pathways, prone to alkylation, may occur. In this review, we will illustrate the chemistry and the biological action of the most studied covalent NLRP3 inhibitors developed so far. A description of what we know about their mechanism of action will address the reader toward a critical understanding of NLRP3 inhibition by electrophilic compounds.

Covalent binders in drug discovery.

Covalent modulation of protein function can have multiple utilities including therapeutics, and probes to interrogate biology. While this field is still viewed with scepticism due to the potential for (idiosyncratic) toxicities, significant strides have been made in terms of understanding how to tune electrophilicity to selectively target specific residues. Progress has also been made in harnessing the potential of covalent binders to uncover novel biology and to provide an enhanced utility as payloads for Antibody Drug Conjugates. This perspective covers the tenets and applications of covalent binders.

Targeted Covalent Inhibitors for Drug Design.

In contrast to the traditional mechanism of drug action that relies on the reversible, noncovalent interaction of a ligand with its biological target, a targeted covalent inhibitor (TCI) is designed such that the initial, reversible association is followed by the formation of a covalent bond between an electrophile on the ligand and a nucleophilic center in the protein. Although this approach offers a variety of potential benefits (high potency and extended duration of action), concerns over the possible toxicological consequences of protein haptenization have hindered the development of the TCI concept. Recently, approaches to mitigate the risk of serious adverse reactions to this new class of agent have emerged, thus stimulating interest in the field and leading to authorization of the first cadre of TCIs to be marketed. The covalent inhibitor approach is rapidly gaining acceptance as a valuable tool in drug discovery, and is poised to make a major impact on the design of enzyme inhibitors and receptor modulators.

Targeting the EGFR/RAS/RAF signaling pathway in anticancer research: a recent update on inhibitor design and clinical trials (2020-2023).

INTRODUCTION: Recent years have seen significant strides in drug developmenttargeting the EGFR/RAS/RAF signaling pathway which is critical forcell growth and proliferation. Protein-protein interaction networksamong EGFR, RAS, and RAF proteins offer insights for drug discovery. This review discusses the drug design and development efforts ofinhibitors targeting these proteins over the past 3 years, detailingtheir structures, selectivity, efficacy, and combination therapy.Strategies to combat drug resistance and minimize toxicities areexplored, along with future research directions. AREA COVERED: This review encompasses clinical trials and patents on EGFR, KRAS,and BRAF inhibitors from 2020 to 2023, including advancements indesign and synthesis of proteolysis targeting chimeras (PROTACs) forprotein degradation. EXPERT OPINION: To tackle drug resistance, designing allosteric fourth-generationEGFR inhibitors is vital. Covalent, allosteric, or combinationaltherapies, along with PROTAC degraders, are key methods to addressresistance and toxicity in KRAS and BRAF inhibitors.

Targeting the PI3K/AKT signaling pathway in anticancer research: a recent update on inhibitor design and clinical trials (2020-2023).

INTRODUCTION: Recent years have witnessed great achievements in drug design and development targeting the phosphatidylinositol 3-kinase/protein kinase-B (PI3K/AKT) signaling pathway, a pathway central to cell growth and proliferation. The nearest neighbor protein-protein interaction networks for PI3K and AKT show the interplays between these target proteins which can be harnessed for drug discovery. In this review, we discuss the drug design and clinical development of inhibitors of PI3K/AKT in the past three years. We review in detail the structures, selectivity, efficacy, and combination therapy of 35 inhibitors targeting these proteins, classified based on the target proteins. Approaches to overcoming drug resistance and to minimizing toxicities are discussed. Future research directions for developing combinational therapy and PROTACs of PI3K and AKT inhibitors are also discussed. AREA COVERED: This review covers clinical trial reports and patent literature on inhibitors of PI3K and AKT published between 2020 and 2023. EXPERT OPINION: To address drug resistance and drug toxicity of inhibitors of PI3K and AKT, it is highly desirable to design and develop subtype-selective PI3K inhibitors or subtype-selective AKT1 inhibitors to minimize toxicity or to develop allosteric drugs that can form covalent bonds. The development of PROTACs of PI3Kα or AKT helps to reduce off-target toxicities.

Structure-property Relationships Reported for the New Drugs Approved in 2023.

Drug-like properties play pivotal roles in drug adsorption, distribution, metabolism, excretion, and toxicity. Therefore, efficiently optimizing these properties is essential for the successful development of novel therapeutics. Understanding the structure-property relationships of clinically approved drugs can provide valuable insights for drug design and optimization strategies. Among the new drugs approved in 2023, which include 31 small-molecule drugs in the US, the structure-property relationships of nine drugs were compiled from the medicinal chemistry literature, in which detailed information on pharmacokinetic and/or physicochemical properties was reported not only for the final drug but also for its key analogs generated during drug development. The structure-property relationships of nine newly approved drugs are summarized, including three kinase inhibitors and three G-protein-coupled receptor antagonists. Several optimization strategies, such as bioisosteric replacement and steric handle installation, have successfully produced clinical candidates with enhanced physicochemical and pharmacokinetic properties. The summarized structure-property relationships demonstrate how appropriate structural modifications can effectively improve overall drug-like properties. The ongoing exploration of structure- property relationships of clinically approved drugs is expected to offer valuable guidance for developing future drugs.

Covalent Modification of Proteins by Osthole Reactive Metabolites using Proteomic Approaches.

BACKGROUND: Osthole (OST) is a bioactive natural coumarin derived from the plant Cnidium monnieri (L.) Cusson fruit (She Chuang Zi), which has various pharmacological and biological activities. OST contains an α,ß- unsaturated lactone, which is an electrophilic group that tends to be metabolized into reactive metabolites (RMs). Then, RMs are able to covalently modify nucleophilic amino acid (AA) residues of target proteins. However, few researchers considered the contribution of the covalent modification induced by OST or its metabolites. OBJECTIVE: This study aims to investigate the metabolic profile and the metabolites-protein modification of OST. METHODS: The metabolites of OST were qualitatively identified using UHPLC-Q-TOF-MS. The RMs modification patterns and potentially modified AA residues were confirmed by UHPLC-Q-TOF-MS using rat liver microsomes (RLMs) and model AAs. Finally, the modified peptides derived from high-abundance microsomal peptides were separated via nano-LC-Orbitrap-MS, and then RM-modified proteins were identified using a proteome discoverer. RESULTS: In the presence of RLMs, OST could rapidly be metabolized within 1 h and hardly identified at 4 h. We detected 10 OST metabolites, 13 OST metabolites-NAC (N-acetyl cysteine) adducts, 3 NAL (N-acetyl lysine) adducts, and 11 GSH (glutathione) adducts. Furthermore, 16 RM-modified protein targets were identified, many of which are included in the essential biological processes of OST's anti-Alzheimer's disease (AD) and anti-tumor. CONCLUSION: This study provides a novel perspective on the molecular mechanism of OST's pharmacological activities, as well as identifies potential targets for further development and application of OST and other Natural products (NPs).

A covalent opsonization approach to enhance synthetic immunity against viral escape variants.

The sensitivity of therapeutic antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral "escape" mutations has inspired efforts to develop treatment strategies that are still effective in the face of rapidly mutating viral surface proteins. Here, we demonstrate a chemical strategy that enforces viral opsonization by natural serum antibodies. This strategy uses chimeric molecules that we call covalent viral opsonizers, which covalently label viral surface proteins, with synthetic antibody-binding ligands. As a proof of concept, we develop covalent viral opsonizers that covalently label the spike protein on SARS-CoV-2 using a "mutation-proof" small-molecule-binding ligand for anti-dinitrophenyl serum antibodies. In model assays, we observe that covalent viral opsonizers can rapidly and selectively covalently label the receptor-binding domain of both native and mutant spike proteins, leading to antibody opsonization. Opsonization mediated by this strategy is able to efficiently block the key binding domain interactions, in contrast to non-covalent analogs. We also show that covalent viral opsonizers enact targeted anti-viral phagocytotic immune function. This strategy has potential general utility for the rapid deployment of anti-viral synthetic immunotherapeutics at the onset of a new pandemic to reinforce vaccination and antibody engineering efforts.

Discovery of Polyphenolic Natural Products as SARS-CoV-2 Mpro Inhibitors for COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the development of direct-acting antiviral drugs due to the coronavirus disease 2019 (COVID-19) pandemic. The main protease of SARS-CoV-2 is a crucial enzyme that breaks down polyproteins synthesized from the viral RNA, making it a validated target for the development of SARS-CoV-2 therapeutics. New chemical phenotypes are frequently discovered in natural goods. In the current study, we used a fluorogenic assay to test a variety of natural products for their ability to inhibit SARS-CoV-2 Mpro. Several compounds were discovered to inhibit Mpro at low micromolar concentrations. It was possible to crystallize robinetin together with SARS-CoV-2 Mpro, and the X-ray structure revealed covalent interaction with the protease's catalytic Cys145 site. Selected potent molecules also exhibited antiviral properties without cytotoxicity. Some of these powerful inhibitors might be utilized as lead compounds for future COVID-19 research.

Quinones as Neuroprotective Agents.

Quinones can in principle be viewed as a double-edged sword in the treatment of neurodegenerative diseases, since they are often cytoprotective but can also be cytotoxic due to covalent and redox modification of biomolecules. Nevertheless, low doses of moderately electrophilic quinones are generally cytoprotective, mainly due to their ability to activate the Keap1/Nrf2 pathway and thus induce the expression of detoxifying enzymes. Some natural quinones have relevant roles in important physiological processes. One of them is coenzyme Q10, which takes part in the oxidative phosphorylation processes involved in cell energy production, as a proton and electron carrier in the mitochondrial respiratory chain, and shows neuroprotective effects relevant to Alzheimer's and Parkinson's diseases. Additional neuroprotective quinones that can be regarded as coenzyme Q10 analogues are idobenone, mitoquinone and plastoquinone. Other endogenous quinones with neuroprotective activities include tocopherol-derived quinones, most notably vatiquinone, and vitamin K. A final group of non-endogenous quinones with neuroprotective activity is discussed, comprising embelin, APX-3330, cannabinoid-derived quinones, asterriquinones and other indolylquinones, pyrroloquinolinequinone and its analogues, geldanamycin and its analogues, rifampicin quinone, memoquin and a number of hybrid structures combining quinones with amino acids, cholinesterase inhibitors and non-steroidal anti-inflammatory drugs.

Extended Applications of Small-Molecule Covalent Inhibitors toward Novel Therapeutic Targets.

Recently, small-molecule covalent inhibitors have been accepted as a practical tool for targeting previously "undruggable" proteins. The high target selectivity of modern covalent inhibitors is now alleviating toxicity concerns regarding the covalent modifications of proteins. However, despite the tremendous clinical success of current covalent inhibitors, there are still unmet medical needs that covalent inhibitors have not yet addressed. This review categorized representative covalent inhibitors based on their mechanism of covalent inhibition: conventional covalent inhibitors, targeted covalent inhibitors (TCIs), and expanded TCIs. By reviewing both Food and Drug Administration (FDA)-approved drugs and drug candidates from recent literature, we provide insight into the future direction of covalent inhibitor development.

Molecular modeling-guided optimization of acetylcholinesterase reactivators: A proof for reactivation of covalently inhibited targets.

Covalent drugs have been intensively studied in some very important fields such as anti-tumor and anti-virus, including the currently global-spread SARS-CoV-2. However, these drugs may interact with a variety of biological macromolecules and cause serious toxicology, so how to reactivate the inhibited targets seems to be imperative in the near future. Organophosphate was an extreme example, which could form a covalent bound easily with acetylcholinesterase and irreversibly inhibited the enzyme, causing high toxicology. Some nucleophilic oxime reactivators for organophosphate poisoned acetylcholinesterase had been developed, but the reactivation process was still less understanding. Herein, we proposed there should be a pre-reactivated pose during the reactivating process and compounds whose binding pose was easy to transfer to the pre-reactivated pose might be efficient reactivators. Then we refined the previous reactivators based on the molecular dynamic simulation results, the resulting compounds L7R3 and L7R5 were proven as much more efficient reactivators for organophosphate inhibited acetylcholinesterase than currently used oximes. This work might provide some insights for constructing reactivators of covalently inhibited targets by using computational methods.

Targeting mutant KRAS.

The protein KRAS has for decades been considered a holy grail of cancer drug discovery. For most of that time, it has also been considered undruggable. Since 2018, five compounds have entered the clinic targeting a single mutant form of KRAS, G12C. Here, we review each of these compounds along with additional approaches to targeting this and other mutants. Remaining challenges include expanding the identification of inhibitors to a broader range of known mutants and to conformations of the protein more likely to avoid development of resistance.