Synthesis, cytotoxicity and antioxidant activity of new conjugates of N-acetyl-d-glucosamine with α-aminophosphonates.

A series of the first conjugates of N-acetyl-d-glucosamine with α-aminophosphonates was synthesized using the Kabachnik-Fields reaction, the Pudovik reaction, a copper(I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) and evaluated for the in vitro cytotoxicity against human cancer cell lines M - HeLa, HuTu-80, A549, PANC-1, MCF-7, T98G and normal lung fibroblast cells WI-38. The tested conjugates, with exception of compound 21b, considered as a lead compound, were either inactive against the used cancer cells or showed moderate cytotoxicity in the range of IC50 values 33-80 µM. The lead compound 21b, being non cytotoxic against normal human cells WI-38 (IC50 = 90 µM), demonstrated good activity (IC50 = 17 µM) against breast adenocarcinoma cells (MCF-7) which to be 1.5 times higher than the activity of the used reference anticancer drug tamoxifen (IC50 = 25.0 µM). A flexible receptor molecular docking simulation showed that the cytotoxicity of the synthesized conjugates of N-acetyl-d-glucosamine with α-aminophosphonates against breast adenocarcinoma MCF-7 cell line is due to their ability to inhibit EGFR kinase domain. In addition, it was found that conjugates 22a and 22b demonstrated antioxidant activity that was not typical for α-aminophosphonates.

Structural basis of human mpox viral DNA replication inhibition by brincidofovir and cidofovir.

Mpox virus has wildly spread over 108 non-endemic regions in the world since May 2022. DNA replication of mpox is performed by DNA polymerase machinery F8-A22-E4, which is known as a great drug target. Brincidofovir and cidofovir are reported to have broad-spectrum antiviral activity against poxviruses, including mpox virus in animal models. However, the molecular mechanism is not understood. Here we report cryogenic electron microscopy structures of mpox viral F8-A22-E4 in complex with a DNA duplex, or dCTP and the DNA duplex, or cidofovir diphosphate and the DNA duplex at resolution of 3.22, 2.98 and 2.79 Å, respectively. Our structural work and DNA replication inhibition assays reveal that cidofovir diphosphate is located at the dCTP binding position with a different conformation to compete with dCTP to incorporate into the DNA and inhibit DNA synthesis. Conformation of both F8-A22-E4 and DNA is changed from the pre-dNTP binding state to DNA synthesizing state after dCTP or cidofovir diphosphate is bound, suggesting a coupling mechanism. This work provides the structural basis of DNA synthesis inhibition by brincidofovir and cidofovir, providing a rational strategy for new therapeutical development for mpox virus and other pox viruses.

Synthetic Methods for Azaheterocyclic Phosphonates and Their Biological Activity: An Update 2004-2024.

The increasing importance of azaheterocyclic phosphonates in the agrochemical, synthetic, and medicinal field has provoked an intense search in the development of synthetic routes for obtaining novel members of this family of compounds. This updated review covers methodologies established since 2004, focusing on the synthesis of azaheterocyclic phosphonates, of which the phosphonate moiety is directly substituted onto to the azaheterocyclic structure. Emphasizing recent advances, this review classifies newly developed synthetic approaches according to the ring size and providing information on biological activities whenever available. Furthermore, this review summarizes information on various methods for the formation of C-P bonds, examining sustainable approaches such as the Michaelis-Arbuzov reaction, the Michaelis-Becker reaction, the Pudovik reaction, the Hirao coupling, and the Kabachnik-Fields reaction. After analyzing the biological activities and applications of azaheterocyclic phosphonates investigated in recent years, a predominant focus on the evaluation of these compounds as anticancer agents is evident. Furthermore, emerging applications underline the versatility and potential of these compounds, highlighting the need for continued research on synthetic methods to expand this interesting family.

Synthesis of Planar Chiral Compounds Containing α-Amino Phosphonates for Antiplant Virus Applications against Potato Virus Y.

An unprecedented study of the application of planar chiral compounds in antiviral pesticide development is reported. A class of multifunctional planar chiral ferrocene derivatives bearing α-amino phosphonate moieties was synthesized. These compounds, exhibiting superior optical purities, were subsequently subjected to antiviral evaluations against the notable plant pathogen potato virus Y (PVY). The influence of the absolute configurations of the planar chiral compounds on their antiviral bioactivities was significant. A number of these enantiomerically enriched planar chiral molecules demonstrated superior anti-PVY activities. Specifically, compound (Sp, R)-9n displayed extraordinary curative activities against PVY, with a 50% maximal effective concentration (EC50) of 216.11 µg/mL, surpassing the efficacy of ningnanmycin (NNM, 272.74 µg/mL). The protective activities of compound (Sp, R)-9n had an EC50 value of 152.78 µg/mL, which was better than that of NNM (413.22 µg/mL). The molecular docking and defense enzyme activity tests were carried out using the planar chiral molecules bearing different absolute configurations to investigate the mechanism of their antiviral activities against PVY. (Sp, R)-9n, (Sp, R)-9o, and NMM all showed stronger affinities to the PVY-CP than the (Rp, S)-9n. Investigations into the mechanisms revealed that the planar chiral configurations of the compounds played pivotal roles in the interactions between the PVY-CP molecules and could augment the activities of the defense enzymes. This study contributes substantial insights into the role of planar chirality in defending plants against viral infections.

Synthesis of LAVR-289, a new [(Z)-3-(acetoxymethyl)-4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid prodrug with pronounced antiviral activity against DNA viruses.

New acyclic pyrimidine nucleoside phosphonate prodrugs with a 4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid skeleton (O-DAPy nucleobase) were prepared through a convergent synthesis by olefin cross-metathesis as the key step. Several acyclic nucleoside 4-(2,4-diaminopyrimidin-6-yl)oxy-but-2-enyl]phosphonic acid prodrug exhibited in vitro antiviral activity in submicromolar or nanomolar range against varicella zoster virus (VZV), human cytomegalovirus (HCMV), human herpes virus type 1 (HSV-1) and type 2 (HSV-2), and vaccinia virus (VV), with good selective index (SI). Among them, the analogue 9c (LAVR-289) proved markedly inhibitory against VZV wild-type (TK+) (EC50 0.0035 µM, SI 740) and for thymidine kinase VZV deficient strains (EC50 0.018 µM, SI 145), with a low morphological toxicity in cell culture at 100 µM and acceptable cytostatic activity resulting in excellent selectivity. Compound 9c exhibited antiviral activity against HCMV (EC50 0.021 µM) and VV (EC50 0.050 µM), as well as against HSV-1 (TK-) (EC50 0.0085 µM). Finally, LAVR-289 (9c) deserves further (pre)clinical investigations as a potent candidate broad-spectrum anti-herpesvirus drug.

N-Functionalization of ß-aminophosphonates: cytotoxic effects of the new derivatives.

ß-Aminophosphonates obtained by the Michael addition of primary amines to the double bond of diethyl vinylphosphonate proved to be suitable starting materials (amine components) in the Kabachnik-Fields reaction with formaldehyde and dialkyl phosphites or secondary phosphine oxides to afford N-phosphonylmethyl- and N-phosphinoylmethyl-ß-aminophosphonates. On the other hand, the starting aminophosphonates were modified by N-acylation using acid chlorides. The N-acyl products were found to exist in a dynamic equilibrium of two conformers as suggested by the broad NMR signals. At 26 °C, there may be rotation around the N-C axis of the acylamide function. At the same time, low-temperature NMR measurements at -5 °C revealed the presence of two distinct rotamers that could be characterized by 31P, 13C and 1H NMR data. The modified ß-aminophosphonic derivatives were subjected to a comparative structure-activity analysis on MDA-MB-231, PC-3, A431 and Ebc-1 tumor cell lines, and in a few cases, significant activity was detected.

Design, synthesis, docking, and antiviral evaluation of some novel pyrimidinone-based α-aminophosphonates as potent H1N1 and HCoV-229E inhibitors.

Dialkyl/aryl aminophosphonates, 3a-g and 4a-e were synthesized using the LiClO4 catalyzed Kabachnic Fields-type reaction straightforwardly and efficiently. The synthesized phosphonates structures were characterized using elemental analyses, FT-IR, 1H NMR, 13C NMR, and MS spectroscopy. The new compounds were subjected to in-silico molecular docking simulations to evaluate their potential inhibition against Influenza A Neuraminidase and RNA-dependent RNA polymerase of human coronavirus 229E. Subsequently, the compounds were further tested in vitro using a cytopathic inhibition assay to assess their antiviral activity against both human Influenza (H1N1) and human coronavirus (HCoV-229E). Diphenyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (furan-2-yl) methyl) phosphonate (3f) and diethyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) methyl) phosphonate (4e) were demonstrated direct inhibition activity against Influenza A Neuraminidase and RNA-dependent RNA polymerase. This was supported by their highly favorable binding energies in-silico, with top-ranked values of -12.5 kcal/mol and -14.2 kcal/mol for compound (3f), and -13.5 kcal/mol and -9.89 kcal/mol for compound (4e). Moreover, they also displayed notable antiviral efficacy in vitro against both viruses. These compounds demonstrated significant antiviral activity, as evidenced by selectivity indices (SI) of 101.7 and 51.8, respectively against H1N1, and 24.5 and 5.1 against HCoV-229E, respectively.

Root Exudates Metabolic Profiling Confirms Distinct Defense Mechanisms Between Cultivars and Treatments with Beneficial Microorganisms and Phosphonate Salts Against Verticillium Wilt in Olive Trees.

Root exudates play a key role in the life cycle of Verticillium dahliae, the causal agent of Verticillium wilt diseases, because they induce microsclerotia germination to initiate plant infection through the roots. In olive plants, the genotype and the application of biological control agents (BCAs) or phosphonate salts influence the ability of root exudates to decrease V. dahliae viability. Understanding the chemical composition of root exudates could provide new insights into the mechanisms of olive plant defense against V. dahliae. Therefore, the main goal of this study was to analyze the metabolomic profiles of root exudates collected from the olive cultivars Arbequina, Frantoio, and Picual subjected to treatment with BCAs (Aureobasidium pullulans AP08, Bacillus amyloliquefaciens PAB-024) or phosphonate salts (copper phosphite, potassium phosphite). These treatments were selected due to their effectiveness as inducers of resistance against Verticillium wilt in olive plants. Our metabolomic analysis revealed that the olive cultivars exhibited differences in root exudates, which could be related to the different degrees of susceptibility to V. dahliae. The composition of root exudates also changed with the application of BCAs or phosphonate fertilizer, highlighting the complex and dynamic nature of the interactions between olive cultivars and treatments preventing V. dahliae infections. Thus, the identification of genotype-specific metabolic changes and specific metabolites induced by these treatments emphasizes the potential of resistance inducers for enhancing plant defense and promoting the growth of beneficial microorganisms.

Oral USC-093, a novel homoserinamide analogue of the tyrosinamide (S)-HPMPA prodrug USC-087 has decreased nephrotoxicity while maintaining antiviral efficacy against human adenovirus infection of Syrian hamsters.

Adenovirus infections of immunocompromised humans are a significant source of morbidity and mortality. Presently, there is no drug specifically approved for the treatment of adenovirus infections by the FDA. The state-of-the-art treatment of such infections is the off-label use of cidofovir, an acyclic nucleotide phosphonate. While cidofovir inhibits adenovirus replication, it has dose-limiting kidney toxicity. There is an apparent need for a better compound to treat adenovirus infections. To this end, we have been developing acyclic nucleotide phosphonate prodrugs that utilize an amino acid scaffold equipped with a lipophilic modifier. Here, we compare the antiviral potential of two prodrugs of HPMPA that differ only in the amino acid-based promoiety: USC-087, based on an N-hexadecyl tyrosinamide, and USC-093, based on an N-hexadecyl serinamide. Oral administration of both compounds was very efficacious against disseminated HAdV-C6 infection in immunosuppressed Syrian hamsters, suppressing virus replication and mitigating pathology even when treatment was withheld until 4 days after challenge. We saw only marginal efficacy after respiratory infection of hamsters, which may reflect suboptimal distribution to the lung. Importantly, neither compound induced intestinal toxicity, which was observed as the major adverse effect in clinical trials of brincidofovir, a prodrug of cidofovir which also contains a C-16 modifier. Notably, we found that there was a significant difference in the nephrotoxicity of the two compounds: USC-087 caused significant kidney toxicity while USC-093 did not, at effective doses. These findings will be valuable guidepoints in the future evolution of this new class of potential prodrugs to treat adenovirus infections.

Discovery of a potent and selective human AC2 inhibitor based on 7-deazapurine analogues of adefovir.

Adefovir based acyclic nucleoside phosphonates were previously shown to modulate bacterial and, to a certain extent, human adenylate cyclases (mACs). In this work, a series of 24 novel 7-substituted 7-deazaadefovir analogues were synthesized in the form of prodrugs. Twelve analogues were single-digit micromolar inhibitors of Bordetella pertussis adenylate cyclase toxin with no cytotoxicity to J774A.1 macrophages. In HEK293 cell-based assays, compound 14 was identified as a potent (IC50 = 4.45 µM), non-toxic, and selective mAC2 inhibitor (vs. mAC1 and mAC5). Such a compound represents a valuable addition to a limited number of small-molecule probes to study the biological functions of individual endogenous mAC isoforms.

Diester Prodrugs of a Phosphonate Butyrophilin Ligand Display Improved Cell Potency, Plasma Stability, and Payload Internalization.

Activation of Vγ9Vδ2 T cells with butyrophilin 3A1 (BTN3A1) agonists such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) has the potential to boost the immune response. Because HMBPP is highly charged and metabolically unstable, prodrugs may be needed to overcome these liabilities, but the prodrugs themselves may be limited by slow payload release or low plasma stability. To identify effective prodrug forms of a phosphonate agonist of BTN3A1, we have prepared a set of diesters bearing one aryl and one acyloxymethyl group. The compounds were evaluated for their ability to stimulate Vγ9Vδ2 T cell proliferation, increase production of interferon γ, resist plasma metabolism, and internalize into leukemia cells. These bioassays have revealed that varied aryl and acyloxymethyl groups can decouple plasma and cellular metabolism and have a significant impact on bioactivity (>200-fold range) and stability (>10 fold range), including some with subnanomolar potency. Our findings increase the understanding of the structure-activity relationships of mixed aryl/acyloxymethyl phosphonate prodrugs.

Phosphonate and Thiasugar Analogues of Glucosamine-6-phosphate: Activation of the glmS Riboswitch and Antibiotic Activity.

The glmS riboswitch is a motif found in 5'-untranslated regions of bacterial mRNA that controls the synthesis of glucosamine-6-phosphate (GlcN6P), an essential building block for the bacterial cell wall, by a feedback mechanism. Activation of the glmS riboswitch by GlcN6P mimics interferes with the ability of bacteria to synthesize its cell wall. Accordingly, GlcN6P mimics acting as glmS activators are promising candidates for future antibiotic drugs that may overcome emerging bacterial resistance against established antibiotics. We describe the synthesis of a series of phosphonate mimics of GlcN6P as well as the thiasugar analogue of GlcN6P. The phosphonate mimics differ in their pKa value to answer the question of whether derivatives with a pKa matching that of GlcN6P would be efficient glmS activators. We found that all derivatives activate the riboswitch, however, less efficiently than GlcN6P. This observation can be explained by the missing hydrogen bonds in the case of phosphonates and is valuable information for the design of future GlcN6P mimics. The thiasugar analogue of GlcN6P on the other hand turned out to be a glmS riboswitch activator with the same activity as the natural metabolite GlcN6P. The nonphosphorylated thiasugar displayed antimicrobial activity against certain bacilli. Therefore, the compound is a promising lead structure for the development of future antibiotics with a potentially novel mode of action.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

Cyclopentenylcytosine (CPE-C): In Vitro and In Vivo Evaluation as an Antiviral against Adenoviral Ocular Infections.

Adenoviruses are the major cause of ocular viral infections worldwide. Currently, there is no approved antiviral treatment for these eye infections. Cyclopentenylcytosine (CPE-C) is an antiviral that has demonstrated activity against more than 20 viruses. The goals of the current study were to determine the in vitro and in vivo antiviral activity of CPE-C as well as its ocular toxicity. Antiviral activity was evaluated in vitro using standard plaque reduction assays to determine the 50% effective concentrations (EC50s) and in vivo in the Ad5/NZW rabbit ocular replication model. Ocular toxicity was determined in uninfected rabbit eyes following topical ocular application. The in vitro EC50s for CPE-C ranged from 0.03 to 0.059 µg/mL for nine adenovirus types that commonly infect the eye. Ocular toxicity testing determined CPE-C to be non-irritating or practically non-irritating by Draize scoring. In vivo, 3% CPE-C topically administered 4X or 2X daily for 7 days to adenovirus-infected eyes demonstrated effective antiviral activity compared with the negative control and comparable antiviral activity to the positive control, 0.5% cidofovir, topically administered twice daily for 7 days. We conclude CPE-C was relatively non-toxic to rabbit eyes and demonstrated potent anti-adenoviral activity in vitro and in vivo.

N2'-Branched Acyclic Nucleoside Phosphonates Containing 9-Deazahypoxanthine as Inhibitors of Plasmodium falciparum 6-Oxopurine Phosphoribosyltransferase.

Twelve N2'-branched acyclic nucleoside phosphonates and bisphosphonates were synthesized as potential inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), the key enzyme in the purine salvage pathway for production of purine nucleotides. The chemical structures were designed with the aim to study selectivity of the inhibitors for PfHGXPRT over human HGPRT. The newly prepared compounds contain 9-deazahypoxanthine connected to a phosphonate group via a five-atom-linker bearing a nitrogen atom (N2') as a branching point. All compounds, with the additional phosphonate group(s) in the second aliphatic linker attached to N2' atom, were low micromolar inhibitors of PfHGXPRT with low to modest selectivity for the parasite enzyme over human HGPRT. The effect of the addition of different chemical groups/linkers to N2' atom on the inhibition constants and selectivity is discussed.

Synthesis, Bioactivity and Molecular Docking of Nereistoxin Derivatives Containing Phosphonate.

Novel nereistoxin derivatives containing phosphonate were synthesized and characterized via 31P, 1H and 13C NMR and HRMS. The anticholinesterase activity of the synthesized compounds was evaluated on human acetylcholinesterase (AChE) using the in vitro Ellman method. Most of the compounds exhibited good inhibition of acetylcholinesterase. All of these compounds were selected to assess their insecticidal activity (in vivo) against Mythimna separata Walker, Myzus persicae Sulzer and Rhopalosiphum padi. Most of the tested compounds displayed potent insecticidal activity against these three species. Compound 7f displayed good activity against all three insect species, showing LC50 values of 136.86 µg/mL for M. separata, 138.37 µg/mL for M. persicae and 131.64 µg/mL for R. padi. Compound 7b had the highest activity against M. persicae and R. padi, with LC50 values of 42.93 µg/mL and 58.19 µg/mL, respectively. Docking studies were performed to speculate the possible binding sites of the compounds and explain the reasons for the activity of the compounds. The results showed that the compounds had lower binding energies with AChE than with the acetylcholine receptor (AchR), suggesting that compounds are more easily bound with AChE.

Enzymatic Synthesis of a Novel Coumarin Aminophosphonates: Antibacterial Effects and Oxidative Stress Modulation on Selected E. coli Strains.

The objective of the present study was to evaluate the synergistic effect of two important pharmacophores, coumarin and α-amino dimethyl phosphonate moieties, on antimicrobial activity toward selected LPS-varied E. coli strains. Studied antimicrobial agents were prepared via a Kabachnik-Fields reaction promoted by lipases. The products were provided with an excellent yield (up to 92%) under mild, solvent- and metal-free conditions. A preliminary exploration of coumarin α-amino dimethyl phosphonate analogs as novel antimicrobial agents was carried out to determine the basic features of the structure responsible for the observed biological activity. The structure-activity relationship revealed that an inhibitory activity of the synthesized compounds is strongly related to the type of the substituents located in the phenyl ring. The collected data demonstrated that coumarin-based α-aminophosphonates can be potential antimicrobial drug candidates, which is particularly crucial due to the constantly increasing resistance of bacteria to commonly used antibiotics.

Optimization of an α-aminonaphthylmethylphosphonic acid inhibitor of purple acid phosphatase using rational structure-based design approaches.

Purple acid phosphatases (PAPs) are ubiquitous binuclear metallohydrolases that have been isolated from various animals, plants and some types of fungi. In humans and mice, elevated PAP activity in osteoclasts is associated with osteoporosis, making human PAP an attractive target for the development of anti-osteoporotic drugs. Based on previous studies focusing on phosphonate scaffolds, as well as a new crystal structure of a PAP in complex with a derivative of a previously synthesized α-aminonaphthylmethylphosphonic acid, phosphonates 24-40 were designed as new PAP inhibitor candidates. Subsequent docking studies predicted that all of these compounds are likely to interact strongly with the active site of human PAP and most are likely to interact strongly with the active site of pig PAP. The seventeen candidates were synthesized with good yields and nine of them (26-28, 30, 33-36 and 38) inhibit in the sub-micromolar to nanomolar range against pig PAP, with 28 and 35 being the most potent mammalian PAP inhibitors reported with Ki values of 168 nM and 186 nM, respectively. This study thus paves the way for the next stage of drug development for phosphonate inhibitors of PAPs as anti-osteoporotic agents.

Possible Interaction between ZnS Nanoparticles and Phosphonates on Mediterranean Clams Ruditapes decussatus.

This study aims to evaluate the toxicity of ZnS nanoparticles (ZnS NP50 = 50 µg/L and ZnS NP100 = 100 µg/L) and diethyl (3-cyano-1-hydroxy-2-methyl-1-phenylpropyl)phosphonate or P (P50 = 50 µg/L and P100 = 100 µg/L) in the clams Ruditapes decussatus using chemical and biochemical approaches. The results demonstrated that clams accumulate ZnS NPs and other metallic elements following exposure. Moreover, ZnS NPs and P separately lead to ROS overproduction, while a mixture of both contaminants has no effect. In addition, data showed that exposure to P100 resulted in increased levels of oxidative stress enzyme activities catalase (CAT) in the gills and digestive glands. A similar trend was also observed in the digestive glands of clams treated with ZnS100. In contrast, CAT activity was decreased in the gills at the same concentration. Exposure to ZnS100 and P100 separately leads to a decrease in acetylcholinesterase (AChE) levels in both gills and digestive glands. Thus, AChE and CAT after co-exposure to an environmental mixture of nanoparticles (ZnS100) and phosphonate (P100) did not show any differences between treated and non-treated clams. The outcome of this work certifies the use of biomarkers and chemical assay when estimating the effects of phosphonate and nanoparticles as part of an ecotoxicological assessment program. An exceptional focus was given to the interaction between ZnS NPs and P. The antioxidant activity of P has been demonstrated to have an additive effect on metal accumulation and antagonistic agents against oxidative stress in clams treated with ZnS NPs.

Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles.

Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification.