Bisubstrate Inhibitors Research Articles

Bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase: Toward cell permeability

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate biosynthesis pathway. It catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). HPPK is essential for microorganisms but is absent in mammals. Yet, it is not the target of any existing antibiotics. Hence, this enzyme is an attractive target for developing novel antimicrobial agents. A wealth of structural and mechanistic information has provided solid basis for structure-based design of HPPK inhibitors. Our bisubstrate inhibitors were initially created by linking 6-hydroxymethylpterin to adenosine through 2, 3, or 4 phosphate groups (HPnA, n = 2, 3, or 4), among which HP4A exhibited the highest binding affinity (Kd = 0.47 ± 0.04 μM). Further development was carried out based on high-resolution structures of HPPK in complex with HP4A. Replacing the phosphate bridge with a piperidine linked thioether eliminated multiple negative charges of the bridge. Substituting the pterin moiety with 7,7-dimethyl-7,8-dihydropterin improved the binding affinity. Arming the piperidine ring with a carboxyl group and oxidizing the thioether further enhanced the potency, resulting in a druglike inhibitor of HPPK (Kd = 0.047 ± 0.007 μM). None of these inhibitors, however, exhibits bacterial cell permeability. It is most likely due to the lack of active folate transporters in bacteria. Replacing the pterin moiety with a 7-deazagaunine moiety, we have obtained a novel bisubstrate inhibitor (HP-101) showing observable cell permeability toward a Gram-positive bacterium. Here, we report the in vitro activity of HP-101 and its structure in complex with HPPK, providing a framework for structure-based further development.

AANAT kinetics of CoASH-targeted electrophiles of tryptamine and related analogs

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the rate-limiting step in melatonin synthesis and is a potential target for disorders involving melatonin overproduction, such as seasonal affective disorder. Previously described AANAT inhibitor bromoacetyltryptamine (BAT) and benzothiophenes analogs were reported to react with CoASH to form potent bisubstrate inhibitors through AANAT’s alkyltransferase function, which is secondary to its role as an acetyltransferase. We replaced the bromoacetyl group in BAT with various Michael acceptors to mitigate possible off-target activity of its bromoacetyl group. Additionally, we modified the length of the carbon linker between the Michael acceptor and indole bicycle of tryptamine to determine its effect on potency. An AANAT enzymatic assay showed a two-carbon linker present in BAT was optimal, while none of the new warheads had activity. Kinetic analysis indicated that these Michael acceptors reacted with CoASH much slower than BAT and not within the timeframe of our enzymatic assay. Additionally, we confirmed earlier reports that the acetyltransferase function of AANAT follows an ordered bi bi mechanism in which AcCoA binds before serotonin. In contrast, BAT’s alkyltransferase kinetics revealed a bi uni mechanism in which BAT binds to AANAT before CoASH. Our model combines both functions of AANAT into one kinetic mechanism.

Development of a chemical scaffold for inhibiting nonribosomal peptide synthetases in live bacterial cells.

The adenylation (A) domain is essential for non-ribosomal peptide synthetases (NRPSs), which synthesize various peptide-based natural products, including virulence factors, such as siderophores and genotoxins. Hence, the inhibition of A-domains could attenuate the virulence of pathogens. 5'-O-N-(Aminoacyl or arylacyl)sulfamoyladenosine (AA-AMS) is a bisubstrate small-molecule inhibitor of the A-domains of NRPSs. However, the bacterial cell permeability of AA-AMS is typically a problem owing to its high hydrophilicity. In this study, we investigated the influence of a modification of 2'-OH in the AMS scaffold with different functional groups on binding to target enzymes and bacterial cell penetration. The inhibitor 7 with a cyanomethyl group at 2'-OH showed desirable inhibitory activity against both recombinant and intracellular gramicidin S synthetase A (GrsA) in the gramicidin S-producer Aneurinibacillus migulanus ATCC 9999, providing an alternative scaffold to develop novel A-domain inhibitors.

Bisubstrate Uridine‐Mimetic‐Peptide Conjugates as O‐GlcNAc Transferase (OGT) Inhibitors

AbstractO‐linked β‐N‐acetylglucosamine transferase (OGT) is the pivotal mammalian enzyme that serves as a nutrient sensor, linking metabolic status to diverse cellular signalling pathways by catalyzing N‐acetylglucosamine transfer to various proteins and detects fluctuations in UDP‐GlcNAc concentrations. Increased UDP‐GlcNAc levels induce O‐GlcNAcylation of proteins, influencing the stability and localization of biomolecules, including transcription factors, kinases, phosphatases, tumour suppressors, and histone‐modifying proteins. Dysregulation and excessive OGT expression can adversely impact cellular processes. The lack of potent and selective inhibitors for OGT has impeded the progress in understanding the enzyme and its molecular mechanisms. This research article details the successful development and synthesis of a series of bisubstrate analogue inhibitors, specifically focusing on inhibiting the O‐GlcNAc transferase (OGT) enzyme. Compound 29 emerged as a promising inhibitor, demonstrating an IC50 value of 134.40 μM in in‐vitro assays. The study emphasizes the crucial role of the acceptor substrate analogue in bisubstrate inhibitor design, highlighting its equal importance alongside the donor substrate analogue. While donor substrate mimetics alone exhibited limited inhibition, their conjugation with appropriate peptides significantly enhanced the overall potency of the final bisubstrate inhibitor. Particularly, peptide derivatives proved superior as acceptor substrate analogues compared to normal substrate peptides. Preliminary data suggest the potential of uridine‐mimetics, like Q6S and Q4 C, following scaffold structure refinement. In‐silico predictions propose that replacing pyrophosphate with a flexible linker substantially improves physicochemical properties relevant to cell permeation. This comprehensive investigation offers valuable insights into designing and optimizing bisubstrate analogue inhibitors for OGT, revealing potential avenues for further refinement and development.

Role and Responsibilities of Various Stakeholders in Pharmacovigilance (PV).

Pharmacovigilance (PV) is the science and practice of monitoring, evaluating, understanding, and preventing adverse drug reactions. Pharmacovigilance (PV) was established by the World Health Organization in response to the thalidomide tragedy of 1961. The main purpose of Pharmacovigilance (PV) is to ensure the safety and efficacy of drugs in clinical practice. Stakeholders: Pharmacovigilance (PV) involves various stakeholders, such as patients, pharmacists, pharmaceutical companies, healthcare professionals, and regulatory authorities. Each stakeholder has a different role and responsibility in reporting, processing, analyzing, and communicating information about adverse drug reactions. Patient engagement is a key factor for enhancing Pharmacovigilance (PV) practices. Pharmacovigilance (PV) relies on data from various sources, such as clinical trials, spontaneous reports, electronic medical records, biomedical literature, and patient-reported data in online health forums. These data sources can provide valuable insights into the real-world use and safety of drugs, as well as the preferences and needs of patients. However, these data sources also pose challenges in terms of quality, validity, reliability, and accessibility. Medicinal Chemistry: Medicinal chemistry is the branch of chemistry that deals with the design, synthesis, and evaluation of new drugs and their biological effects. Medicinal chemistry can enhance Pharmacovigilance (PV) practices by finding new therapeutic indications for existing drugs or compounds that have already been tested for safety and efficacy. Medicinal chemistry also requires careful design and evaluation of covalent inhibitors, bi-substrate inhibitors, stabilizers of protein non-effective conformations, and hydrophobic pocket modifiers to ensure their safety and efficacy. Pharmacovigilance (PV) is a dynamic and evolving discipline that requires collaboration, regulation, education, and innovation to improve patient safety and care. This review aims to provide a comprehensive overview of the potential and issues associated with Pharmacovigilance (PV) practices.

Comparative Analysis of Two NNMT Bisubstrate Inhibitors through Chemoproteomic Studies: Uncovering the Role of Unconventional SAM Analogue Moiety for Improved Selectivity.

Unconventional S-adenosyl-L-methionine (SAM) mimics with enhanced hydrophobicity are an adaptable building block to develop cell-potent inhibitors for SAM-dependent methyltransferases as targeted therapeutics. We recently discovered cell-potent bisubstrate inhibitors for nicotinamide N-methyltransferase (NNMT) by using an unconventional SAM mimic. To delve into the selectivity implications of the unconventional SAM mimic, we employed a chemoproteomic approach to assess two potent NNMT inhibitors LL320 (Ki,app = 6.8 nM) and II399 (containing an unconventional SAM mimic, Ki,app = 5.9 nM) within endogenous proteomes. Our work began with the rational design and synthesis of immobilized probes 1 and 2, utilizing LL320 and II399 as parent compounds. Systematic analysis of protein networks associated with these probes revealed a comprehensive landscape. Notably, NNMT emerged as the top-ranking hit, substantiating the high selectivity of both inhibitors. Meanwhile, we identified additional interacting proteins for LL320 (38) and II399 (17), showcasing the intricate selectivity profiles associated with these compounds. Subsequent experiments confirmed LL320's interactions with RNMT, DPH5, and SAHH, while II399 exhibited interactions with SHMT2 and MEPCE. Importantly, incorporating the unconventional SAM mimic in II399 led to improved selectivity compared to LL320. Our findings underscore the importance of selectivity profiling and validate the utilization of the unconventional SAM mimic as a viable strategy to create highly selective and cell-permeable inhibitors for SAM-dependent methyltransferases.

Structure-guided design of a selective inhibitor of the methyltransferase KMT9 with cellular activity.

Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved incell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.

5′-cap RNA/SAM mimetic conjugates as bisubstrate inhibitors of viral RNA cap 2′-O-methyltransferases

Viral RNA cap 2′-O-methyltransferases are considered promising therapeutic targets for antiviral treatments, as they play a key role in the formation of viral RNA cap-1 structures to escape the host immune system. A better understanding of how they interact with their natural substrates (RNA and the methyl donor SAM) would enable the rational development of potent inhibitors. However, as few structures of 2′-O-MTases in complex with RNA have been described, little is known about substrate recognition by these MTases. For this, chemical tools mimicking the state in which the cap RNA substrate and SAM cofactor are bound in the enzyme’s catalytic pocket may prove useful. In this work, we designed and synthesized over 30 RNA conjugates that contain a short oligoribonucleotide (ORN with 4 or 6 nucleotides) with the first nucleotide 2′-O-attached to an adenosine by linkers of different lengths and containing S or N-heteroatoms, or a 1,2,3-triazole ring. These ORN conjugates bearing or not a cap structure at 5′-extremity mimic the methylation transition state with RNA substrate/SAM complex as bisubstrates of 2′-O-MTases. The ORN conjugates were synthesized either by the incorporation of a dinucleoside phosphoramidite during RNA elongation or by click chemistry performed on solid-phase post-RNA elongation. Their ability to inhibit the activity of the nsp16/nsp10 complex of SARS-CoV-2 and the NS5 protein of dengue and Zika viruses was assessed. Significant submicromolar IC50 values and Kd values in the µM range were found, suggesting a possible interaction of some ORN conjugates with these viral 2′-O-MTases.

Enhancing nicotinamide N-methyltransferase bisubstrate inhibitor activity through 7-deazaadenosine and linker modifications

Nicotinamide N-methyltransferase (NNMT) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to nicotinamide (NAM) and other pyridine-related compounds and is involved in various metabolic processes in the human body. In addition, abnormal expression of NNMT occurs under various pathological conditions such as cancer, diabetes, metabolic disorders, and neurodegenerative diseases, making it a promising drug target worthy of in-depth research. Small-molecule NNMT inhibitors with high potency and selectivity are necessary chemical tools to test biological hypotheses and potential therapies. In this study, we developed a series of highly active NNMT inhibitors by modifying N7 position of adenine. Among them, compound 3–12 (IC50 = 47.9 ± 0.6 nM) exhibited potent inhibitory activity and also had an excellent selectivity profile over a panel of human methyltransferases. We showed that the N7 position of adenine in the NNMT bisubstrate inhibitor was a modifiable site, thus offering insights into the development of NNMT inhibitors.

Structure-guided design and synthesis of ATP-competitive N-acyl-substituted sulfamide d-alanine-d-alanine ligase inhibitors

d-Alanine-d-alanine ligase (Ddl) catalyses the ATP-dependent formation of d-Ala-d-Ala, a critical component in bacterial cell wall biosynthesis and is a validated target for new antimicrobial agents. Here, we describe the structure-guided design, synthesis, and evaluation of ATP-competitive N-acyl-substituted sulfamides 27–36, 42, 46, 47 as inhibitors of Staphylococcus aureus Ddl (SaDdl). A crystal structure of SaDdl complexed with ATP and d-Ala-d-Ala (PDB: 7U9K) identified ATP-mimetic 8 as an initial scaffold for further inhibitor design. Evaluation of 8 in SaDdl enzyme inhibition assays revealed the ability to reduce enzyme activity to 72 ± 8 % (IC50 = 1.6 mM). The sulfamide linker of 8 was extended with 2-(4-methoxyphenyl)ethanol to give 29, to investigate further interactions with the d-Ala pocket of SaDdl, as predicted by molecular docking. This compound reduced enzyme activity to 89 ± 1 %, with replacement of the 4-methoxyphenyl group in 29 with alternative phenyl substituents (27, 28, 31–33, 35, 36) failing to significantly improve on this (80–89 % remaining enzyme activity). Exchanging these phenyl substituents with selected heterocycles (42, 46, 47) did improve activity, with the most active compound (42) reducing SaDdl activity to 70 ± 1 % (IC50 = 1.7 mM), which compares favourably to the FDA-approved inhibitor d-cycloserine (DCS) (IC50 = 0.1 mM). To the best of our knowledge, this is the first reported study of bisubstrate SaDdl inhibitors.

Structure-Activity Relationship Studies on Cell-Potent Nicotinamide N-Methyltransferase Bisubstrate Inhibitors.

Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme implicated in multiple diseases, making it a promising therapeutic target. Building upon our recently reported NNMT inhibitor II399, we systematically investigate the structure-activity relationship by designing and synthesizing a series of analogues. Among them, two top inhibitors II559 (Ki = 1.2 nM) and II802 (Ki = 1.6 nM) displayed over 5000-fold selectivity for NNMT over closely related methyltransferases. Moreover, II559 and II802 showed enhanced cellular inhibition, with a cellular IC50 value of approximately 150 nM, making them the most cell-potent bisubstrate inhibitors reported to date. Furthermore, both inhibitors reduced the cell viability with a GI50 value of ∼10 μM and suppressed the migration of aggressive clear cell renal cancer cell carcinoma cell lines. Overall, II559 and II802 would serve as valuable probes to investigate the enzymatic function of NNMT in health and diseases.

Structures of SARS-CoV-2 N7-methyltransferase with DOT1L and PRMT7 inhibitors provide a platform for new antivirals.

The RNA N7-methyltransferase (MTase) activity of SARS-CoV-2's nsp14 protein is essential for viral replication and is a target for the development of new antivirals. Nsp14 uses S-adenosyl methionine (SAM) as the methyl donor to cap the 5' end of the SARS-CoV-2 mRNA and generates S-adenosyl homocysteine (SAH) as the reaction byproduct. Due to the central role of histone MTases in cancer, many SAM/SAH analogs with properties of cell permeability have recently been developed for the inhibition of these MTases. We have succeeded in identifying two such compounds (SGC0946 and SGC8158) that display significant antiviral activity and bind to the SARS-CoV-2 nsp14 N7-MTase core. Unexpectedly, crystal structures of SGC0946 and SGC8158 with the SARS-CoV-2 nsp14 N7-MTase core identify them as bi-substrate inhibitors of the viral MTase, co-occupying both the SAM and RNA binding sites; positing novel features that can be derivatized for increased potency and selectivity for SARS-CoV-2 nsp14. Taken together, the high-resolution structures and the accompanying biophysical and viral replication data provide a new avenue for developing analogs of SGC0946 and SGC8158 as antivirals.

Optimized bisubstrate inhibitors for the actin N-terminal acetyltransferase NAA80.

Acetylation of protein N-termini is one of the most common protein modifications in the eukaryotic cell and is catalyzed by the N-terminal acetyltransferase family of enzymes. The N-terminal acetyltransferase NAA80 is expressed in the animal kingdom and was recently found to specifically N-terminally acetylate actin, which is the main component of the microfilament system. This unique animal cell actin processing is essential for the maintenance of cell integrity and motility. Actin is the only known substrate of NAA80, thus potent inhibitors of NAA80 could prove as important tool compounds to study the crucial roles of actin and how NAA80 regulates this by N-terminal acetylation. Herein we describe a systematic study toward optimizing the peptide part of a bisubstrate-based NAA80 inhibitor comprising of coenzyme A conjugated onto the N-terminus of a tetrapeptide amide via an acetyl linker. By testing various combinations of Asp and Glu which are found at the N-termini of β- and γ-actin, respectively, CoA-Ac-EDDI-NH2 was identified as the best inhibitor with an IC50 value of 120nM.

Semi-Synthetic CoA-α-Synuclein Constructs Trap N-Terminal Acetyltransferase NatB for Binding Mechanism Studies.

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases. A major member of this enzyme family, NatB, acts on much of the human proteome, including α-synuclein (αS), a synaptic protein that mediates vesicle trafficking. NatB acetylation of αS modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinson's disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of αS have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here, we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human αS, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, αS remains disordered when in complex with hNatB. We further probe changes in the αS conformation by single molecule Förster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes as well as their implications for hNatB substrate recognition and specific inhibition of the interaction with αS. Beyond the study of αS and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling.

Plasmodium falciparum Eukaryotic Translation Initiation Factor 3 is Stabilized by Quinazoline-Quinoline Bisubstrate Inhibitors.

Malaria drug resistance is hampering the fight against the deadliest parasitic disease affecting over 200 million people worldwide. We recently developed quinoline-quinazoline-based inhibitors (as compound 70) as promising new antimalarials. Here, we aimed to investigate their mode of action by using thermal proteome profiling (TPP). The eukaryotic translation initiation factor 3 (EIF3i) subunit I was identified as the main target protein stabilized by compound 70 in Plasmodium falciparum. This protein has never been characterized in malaria parasites. P. falciparum parasite lines were generated expressing either a HA tag or an inducible knockdown of the PfEIF3i gene to further characterize the target protein. PfEIF3i was stabilized in the presence of compound 70 in a cellular thermal shift Western blot assay, pointing that PfEIF3i indeed interacts with quinoline-quinazoline-based inhibitors. In addition, PfEIF3i-inducible knockdown blocks intra-erythrocytic development in the trophozoite stage, indicating that it has a vital function. We show that PfEIF3i is mostly expressed in late intra-erythrocytic stages and localizes in the cytoplasm. Previous mass spectrometry reports show that PfEIF3i is expressed in all parasite life cycle stages. Further studies will explore the potential of PfEIF3i as a target for the design of new antimalarial drugs active all along the life cycle of the parasite.

Understanding HAT1: A Comprehensive Review of Noncanonical Roles and Connection with Disease.

Histone acetylation plays a vital role in organizing chromatin, regulating gene expression and controlling the cell cycle. The first histone acetyltransferase to be identified was histone acetyltransferase 1 (HAT1), but it remains one of the least understood acetyltransferases. HAT1 catalyzes the acetylation of newly synthesized H4 and, to a lesser extent, H2A in the cytoplasm. However, 20 min after assembly, histones lose acetylation marks. Moreover, new noncanonical functions have been described for HAT1, revealing its complexity and complicating the understanding of its functions. Recently discovered roles include facilitating the translocation of the H3H4 dimer into the nucleus, increasing the stability of the DNA replication fork, replication-coupled chromatin assembly, coordination of histone production, DNA damage repair, telomeric silencing, epigenetic regulation of nuclear lamina-associated heterochromatin, regulation of the NF-κB response, succinyl transferase activity and mitochondrial protein acetylation. In addition, the functions and expression levels of HAT1 have been linked to many diseases, such as many types of cancer, viral infections (hepatitis B virus, human immunodeficiency virus and viperin synthesis) and inflammatory diseases (chronic obstructive pulmonary disease, atherosclerosis and ischemic stroke). The collective data reveal that HAT1 is a promising therapeutic target, and novel therapeutic approaches, such as RNA interference and the use of aptamers, bisubstrate inhibitors and small-molecule inhibitors, are being evaluated at the preclinical level.

Structure-Based Design of Bisubstrate Tetracycline Destructase Inhibitors That Block Flavin Redox Cycling.

Tetracyclines (TCs) are an important class of antibiotics threatened by an emerging new resistance mechanism─enzymatic inactivation. These TC-inactivating enzymes, also known as tetracycline destructases (TDases), inactivate all known TC antibiotics, including drugs of last resort. Combination therapies consisting of a TDase inhibitor and a TC antibiotic represent an attractive strategy for overcoming this type of antibiotic resistance. Here, we report the structure-based design, synthesis, and evaluation of bifunctional TDase inhibitors derived from anhydrotetracycline (aTC). By appending a nicotinamide isostere to the C9 position of the aTC D-ring, we generated bisubstrate TDase inhibitors. The bisubstrate inhibitors have extended interactions with TDases by spanning both the TC and presumed NADPH binding pockets. This simultaneously blocks TC binding and the reduction of FAD by NADPH while "locking" TDases in an unproductive FAD "out" conformation.

3-(Adenosylthio)benzoic Acid Derivatives as SARS-CoV-2 Nsp14 Methyltransferase Inhibitors

SARS-CoV-2 nsp14 guanine-N7-methyltransferase plays an important role in the viral RNA translation process by catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to viral mRNA cap. We report a structure-guided design and synthesis of 3-(adenosylthio)benzoic acid derivatives as nsp14 methyltransferase inhibitors resulting in compound 5p with subnanomolar inhibitory activity and improved cell membrane permeability in comparison with the parent inhibitor. Compound 5p acts as a bisubstrate inhibitor targeting both SAM and mRNA-binding pockets of nsp14. While the selectivity of 3-(adenosylthio)benzoic acid derivatives against human glycine N-methyltransferase was not improved, the discovery of phenyl-substituted analogs 5p,t may contribute to further development of SARS-CoV-2 nsp14 bisubstrate inhibitors.

Over 40 Years of Fosmidomycin Drug Research: A Comprehensive Review and Future Opportunities.

To address the continued rise of multi-drug-resistant microorganisms, the development of novel drugs with new modes of action is urgently required. While humans biosynthesize the essential isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the established mevalonate pathway, pathogenic protozoa and certain pathogenic eubacteria use the less well-known methylerythritol phosphate pathway for this purpose. Important pathogens using the MEP pathway are, for example, Plasmodium falciparum, Mycobacterium tuberculosis, Pseudomonas aeruginosa and Escherichia coli. The enzymes of that pathway are targets for antiinfective drugs that are exempt from target-related toxicity. 2C-Methyl-D-erythritol 4-phosphate (MEP), the second enzyme of the non-mevalonate pathway, has been established as the molecular target of fosmidomycin, an antibiotic that has so far failed to be approved as an anti-infective drug. This review describes the development and anti-infective properties of a wide range of fosmidomycin derivatives synthesized over the last four decades. Here we discuss the DXR inhibitor pharmacophore, which comprises a metal-binding group, a phosphate or phosphonate moiety and a connecting linker. Furthermore, non-fosmidomycin-based DXRi, bisubstrate inhibitors and several prodrug concepts are described. A comprehensive structure-activity relationship (SAR) of nearly all inhibitor types is presented and some novel opportunities for further drug development of DXR inhibitors are discussed.

Deactivatable Bisubstrate Inhibitors of Protein Kinases.

Bivalent ligands, including bisubstrate inhibitors, are conjugates of pharmacophores, which simultaneously target two binding sites of the biomolecule. Such structures offer attainable means for the development of compounds whose ability to bind to the biological target could be modulated by an external trigger. In the present work, two deactivatable bisubstrate inhibitors of basophilic protein kinases (PKs) were constructed by conjugating the pharmacophores via linkers that could be cleaved in response to external stimuli. The inhibitor ARC-2121 incorporated a photocleavable nitrodibenzofuran-comprising β-amino acid residue in the structure of the linker. The pharmacophores of the other deactivatable inhibitor ARC-2194 were conjugated via reduction-cleavable disulfide bond. The disassembly of the inhibitors was monitored by HPLC-MS. The affinity and inhibitory potency of the inhibitors toward cAMP-dependent PK (PKAcα) were established by an equilibrium competitive displacement assay and enzyme activity assay, respectively. The deactivatable inhibitors possessed remarkably high 1–2-picomolar affinity toward PKAcα. Irradiation of ARC-2121 with 365 nm UV radiation led to reaction products possessing a 30-fold reduced affinity. The chemical reduction of ARC-2194 resulted in the decrease of affinity of over four orders of magnitude. The deactivatable inhibitors of PKs are valuable tools for the temporal inhibition or capture of these pharmacologically important enzymes.