Development of the First Potential Nonpeptidic Positron Emission Tomography Tracer for the Imaging of CCR2 Receptors

Herein we report the design and synthesis of a series of highly selective CCR2 antagonists as ¹⁸F-labeled PET tracers. The derivatives were evaluated extensively for their off-target profile at 48 different targets. The most potent and selective candidate was applied in vivo in a biodistribution study, demonstrating a promising profile for further preclinical development. This compound represents the first potential nonpeptidic PET tracer for the imaging of CCR2 receptors.     

A Novel Way To Radiolabel Human Butyrylcholinesterase for Positron Emission Tomography through Irreversible Transfer of the Radiolabeled Moiety

The enzyme butyrylcholinesterase (BChE) is known to be involved in the detoxification of xenobiotics in blood plasma and is associated with the progress of neurodegenerative disorders, diabetes type 2, obesity, and diseases of the cardiovascular system. In the present study, we developed carbamate‐based inhibitors serving as positron emission tomography (PET) radiotracers with ¹⁸F and ¹¹C as radioisotopes to visualize BChE distribution. These inhibitors are radiolabeled at the carbamate site and transfer this moiety onto BChE, which thus results in covalent and permanent radiolabeling of the enzyme. There are no comparable radiotracers for cholinesterases described to date. By ex vivo autoradiography experiments on mice brain slices and kinetic investigations, selective and covalent transfer of the radiolabeled carbamate moiety onto BChE was proven. These tracers might provide high resolution of BChE distribution in vivo to enable investigations into the pathophysiological mechanisms of diseases associated with alterations in BChE occurrence.     

4,4,16-Trifluoropalmitate: Design,Synthesis, Tritiation,Radiofluorination and Preclinical PET Imaging Studies on Myocardial Fatty Acid Oxidation

Fatty acid oxidation (FAO) produces most of the ATP used to sustain the cardiac contractile work, although glycolysis is a secondary source of ATP under normal physiological conditions. FAO impairment has been reported in the advanced stages of heart failure (HF) and is strongly linked to disease progression and severity. Thus, from a clinical perspective, FAO dysregulation provides prognostic value for HF progression, the assessment of which could be used to improve patient monitoring and the effectiveness of therapy. Positron emission tomography (PET) imaging represents a powerful tool for the assessment and quantification of metabolic pathways in vivo. Several FAO PET tracers have been reported in the literature, but none of them is in routine clinical use yet. Metabolically trapped tracers are particularly interesting because they undergo FAO to generate a radioactive metabolite that is subsequently trapped in the mitochondria, thus providing a quantitative means of measuring FAO in vivo. Herein, we describe the design, synthesis, tritium labelling and radiofluorination of 4,4,16-trifluoro-palmitate ( 1 ) as a novel potential metabolically trapped FAO tracer. Preliminary PET-CT studies on [¹⁸F] 1 in rats showed rapid blood clearance, good metabolic stability – confirmed by using [³H] 1 in vitro – and resistance towards defluorination. However, cardiac uptake in rats was modest (0.24±0.04 % ID/g), and kinetic analysis showed reversible uptake, thus indicating that [¹⁸F] 1 is not irreversibly trapped.     

Mapping the Binding Interface of PET Tracer Molecules and Alzheimer Disease Aβ Fibrils by Using MAS Solid-State NMR Spectroscopy

Positron emission tomography (PET) tracer molecules like thioflavin T specifically recognize amyloid deposition in brain tissue by selective binding to hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The molecular basis of this interaction is, however, not well understood. We have employed magic angle spinning (MAS) solid-state NMR spectroscopy to characterize Aβ-PET tracer complexes at atomic resolution. We established a titration protocol by using bovine serum albumin as a carrier to transfer hydrophobic small molecules to Aβ(1-40) fibrillar aggregates. The same Aβ(1-40) amyloid fibril sample was employed in subsequent titrations to minimize systematic errors that potentially arise from sample preparation. In the experiments, the small molecules ¹³C-methylated Pittsburgh compound B (PiB) as well as a novel Aβ tracer based on a diarylbithiazole (DABTA) scaffold were employed. Classical ¹³C-detected as well as proton-detected spectra of protonated and perdeuterated samples with back-substituted protons, respectively, were acquired and analyzed. After titration of the tracers, chemical-shift perturbations were observed in the loop region involving residues Gly25-Lys28 and Ile32-Gly33, thus suggesting that the PET tracer molecules interact with the loop region connecting β-sheets β1 and β2 in Aβ fibrils. We found that titration of the PiB derivatives suppressed fibril polymorphism and stabilized the amyloid fibril structure.     

Evaluation of 5H-Thiazolo[3,2-α]pyrimidin-5-ones as Potential GluN2A PET Tracers

We describe here our efforts to develop a PET tracer for imaging GluN2A-containing NMDA receptors, based on a 5H-thiazolo[3,2-α]pyrimidin-5-one scaffold. The metabolic stability and overall properties could be optimized satisfactorily, although binding affinities remained a limiting factor for in vivo imaging. We nevertheless identified 7-(((2-fluoroethyl)(3-fluorophenyl)amino)-methyl)-3-(2-(hydroxymethyl)cyclopropyl)-2-methyl-5H-thiazolo-[3,2-α]pyrimidin-5-one ([¹⁸F] 7b ) as a radioligand providing good-quality images in autoradiographic studies, as well as a tritiated derivative, 2-(7-(((2-fluoroethyl)(4-fluorophenyl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-α]pyrimidin-3-yl)cyclopropane-1-carbonitrile ([³H₂] 15b ), which was used for the successful development of a radioligand binding assay. These are valuable new tools for the study of GluN2A-containing NMDA receptors, and for the optimization of allosteric modulators binding to the pharmacophore located at the dimer interface of the GluN1-GluN2A ligand-binding domain.     

In vivo 13C nuclear magnetic resonance: Applications and current limitations for noninvasive assessment of fatty acid status

As a noninvasive method,in vivo ¹³C nuclear magnetic resonance has potentially important applications in understanding the metabolism of long chain fatty acids in organs of living humans. At present, this methodology is most advanced for research on glucose utilization. However, the main¹³C signals visiblein vivo are from fatty acids in adipose tissue and the olefinic signals can be used to noninvasively estimate adipose tissue content and relative dietary intake of polyunsaturates and monounsaturates. The low natural abundance of¹³C improves the utility of this isotope for fatty acid tracer studies. Due to excessive signal broadening, uniform¹³C-labelling seems to have limited application inin vivo fatty acid studies. Tracer fatty acids with¹³C enrichment at a specific carbon position, i.e., [3-¹³C]_γ-linolenate, appear to be the most useful forin vivo tracer studies. Development of methods permitting resolution of¹³C enrichment in structural lipids of lean tissues will be an important breakthrough which may make human tracer studies feasible and worthwhile.     

Comparative Biochemistry of Four Polyester (PET) Hydrolases**

The potential of bioprocessing in a circular plastic economy has strongly stimulated research into the enzymatic degradation of different synthetic polymers. Particular interest has been devoted to the commonly used polyester, poly(ethylene terephthalate) (PET), and a number of PET hydrolases have been described. However, a kinetic framework for comparisons of PET hydrolases (or other plastic-degrading enzymes) acting on the insoluble substrate has not been established. Herein, we propose such a framework, which we have tested against kinetic measurements for four PET hydrolases. The analysis provided values of k_cat and K_M, as well as an apparent specificity constant in the conventional units of M⁻¹s⁻¹. These parameters, together with experimental values for the number of enzyme attack sites on the PET surface, enabled comparative analyses. A variant of the PET hydrolase from Ideonella sakaiensis was the most efficient enzyme at ambient conditions; it relied on a high k_cat rather than a low K_M. Moreover, both soluble and insoluble PET fragments were consistently hydrolyzed much faster than intact PET. This suggests that interactions between polymer strands slow down PET degradation, whereas the chemical steps of catalysis and the low accessibility associated with solid substrate were less important for the overall rate. Finally, the investigated enzymes showed a remarkable substrate affinity, and reached half the saturation rate on PET when the concentration of attack sites in the suspension was only about 50 nM. We propose that this is linked to nonspecific adsorption, which promotes the nearness of enzyme and attack sites.     

Cover Feature: A Comparative Study of in vitro Assays for Predicting the Nonspecific Binding of PET Imaging Agents in vivo (ChemMedChem 7/2020)

Nonspecific binding (NSB) is a key parameter in optimizing PET imaging tracers. We compared the ability to predict NSB of three available methods: LIMBA, rat f_u,brain, and CHI(IAM). Even though NSB is often associated with lipophilicity, we observed that logD does not correlate with any of these assays, clearly indicating that lipophilicity, while influencing NSB, is insufficient to predict it. A cross-comparison of the methods showed that all three correlate and are useful predictors of NSB. The three assays, however, rank the molecules slightly differently, illustrating the challenge of comparing molecules within a narrow chemical space. We also noted that CHI(IAM) values more effectively predict V_NS, a measure of in vivo NSB in the human brain. CHI(IAM) measurements might be a closer model of the actual physicochemical interaction between PET tracer candidates and cell membranes, and seems to be the method of choice for the optimization of in vivo NSB.     

Conjugates of Tacrine with Salicylamide as Promising Multitarget Agents for Alzheimer's Disease

New conjugates of tacrine and salicylamide with alkylene spacers were synthesized and evaluated as potential multifunctional agents for Alzheimer's disease (AD). The compounds exhibited high acetylcholinesterase (AChE, IC₅₀ to 0.224 μM) and butyrylcholinesterase (BChE, IC₅₀ to 0.0104 μM) inhibitory activities. They were also rather poor inhibitors of carboxylesterase, suggesting a low tendency to exert potential unwanted drug-drug interactions in clinical use. The conjugates were mixed-type reversible inhibitors of both cholinesterases and demonstrated dual binding to the catalytic and peripheral anionic sites of AChE in molecular docking that, along with experimental results on propidium iodide displacement, suggest their potential to block AChE-induced β-amyloid aggregation. The new conjugates exhibited high ABTS^.+-scavenging activity. N-(6-(1,2,3,4-Tetrahydroacridin-9-ylamino)hexyl)salicylamide is a lead compound that also demonstrates metal chelating ability toward Cu²⁺, Fe²⁺ and Zn²⁺. Thus, the new conjugates have displayed the potential to be multifunctional anti-AD agents for further development.     

Design,Synthesis, Radiosynthesis and Biological Evaluation of Fenretinide Analogues as Anticancer and Metabolic Syndrome-Preventive Agents

Fenretinide (4-HPR) is a synthetic derivative of all-trans-retinoic acid (ATRA) characterised by improved therapeutic properties and toxicological profile relative to ATRA. 4-HPR has been mostly investigated as an anti-cancer agent, but recent studies showed its promising therapeutic potential for preventing metabolic syndrome. Several biological targets are involved in 4-HPR's activity, leading to the potential use of this molecule for treating different pathologies. However, although 4-HPR displays quite well-understood multitarget promiscuity with regards to pharmacology, interpreting its precise physiological role remains challenging. In addition, despite promising results in vitro, the clinical efficacy of 4-HPR as a chemotherapeutic agent has not been satisfactory so far. Herein, we describe the preparation of a library of 4-HPR analogues, followed by the biological evaluation of their anti-cancer and anti-obesity/diabetic properties. The click-type analogue 3 b showed good capacity to reduce the amount of lipid accumulation in 3T3-L1 adipocytes during differentiation. Furthermore, it showed an IC₅₀ of 0.53±0.8 μM in cell viability tests on breast cancer cell line MCF-7, together with a good selectivity (SI=121) over noncancerous HEK293 cells. Thus, 3 b was selected as a potential PET tracer to study retinoids in vivo, and the radiosynthesis of [¹⁸F] 3b was successfully developed. Unfortunately, the stability of [¹⁸F] 3b turned out to be insufficient to pursue imaging studies.     

Synthesis and Pharmacological Evaluation of Fluorinated Quinoxaline-Based κ-Opioid Receptor (KOR) Agonists Designed for PET Studies

κ-Opioid receptors (KORs) play a predominant role in pain alleviation, itching skin diseases, depression and neurodegenerative disorders such as multiple sclerosis. Therefore, imaging of KOR by a fluorinated PET tracer was envisaged. Two strategies were followed to introduce a F atom into the very potent class of cis,trans-configured perhydroquinoxalines. Whereas the synthesis of fluoroethyltriazole 2 has already been reported, fluoropyrrolidines 14 (1-[2-(3,4-dichlorophenyl)acetyl]-8-[(R)-3-fluoropyrrolidin-1-yl]-perhydroquinoxalines) were prepared by S_N2 substitution of a cyclic sulfuric acid derivative with hydroxypyrrolidine and subsequent transformation of the OH moiety into a F substituent. Fluoropyrrolidines 14 showed similar low-nanomolar KOR affinity and selectivity to the corresponding pyrrolidines, but the corresponding alcohols were slightly less active. In the cAMP and β-arrestin assay, 14b (proton at the 4-position) exhibited similar KOR agonistic activity as U-50,488. The fluoro derivatives 14b and 14c (CO₂CH₃ at the 4-position) revealed KOR-mediated anti-inflammatory activity as CD11c and the IFN-γ production were reduced significantly in mouse and human dendritic cells. Compounds 14b and 14-c also displayed anti-inflammatory and immunomodulatory activity in mouse and human T cells. The PET tracer [¹⁸F]- 2 was prepared by 1,3-dipolar cycloaddition. In vivo, [¹⁸F]- 2 did not label KOR due to very fast elimination kinetics. Nucleophilic substitution of a mesylate precursor provided [¹⁸F]- 14c . Unfortunately, defluorination of [¹⁸F]- 14c occurred in vivo, which was analyzed in detail by in vitro studies.     

MS565: A SPECT Tracer for Evaluating the Brain Penetration of BAF312 (Siponimod)

BAF312 (siponimod) is a sphingosine‐1‐phosphate (S1P) receptor modulator in clinical development for the treatment of multiple sclerosis, with faster organ/tissue distribution and elimination kinetics than its precursor FTY720 (fingolimod). Our aim was to develop a tracer to better quantify the penetration of BAF312 in the human brain, with the potential to be labeled for positron emission tomography (PET) or single‐photon emission computed tomography (SPECT). Although the PET radioisotopes ¹¹C and ¹⁸F could have been introduced in BAF312 without modifying its structure, they do not have decay kinetics compatible with the time required for observing the drug′s organ distribution in patients. In contrast, the SPECT radioisotope ¹²³I has a longer half‐life and would suit this purpose. Herein we report the identification of an iodinated derivative of BAF312, (E)‐1‐(4‐(1‐(((4‐cyclohexyl‐3‐iodobenzyl)oxy)imino)ethyl)‐2‐ethylbenzyl)azetidine‐3‐carboxylic acid ( 18 , MS565), as a SPECT tracer candidate with affinity, S1P receptor selectivity, overall physicochemical properties, and blood pharmacokinetics similar to those of the original molecule. A whole‐body autoradiography study performed with [¹⁴C]MS565 subsequently confirmed that its organ distribution is similar to that of BAF312. This validates the selection of MS565 for ¹²³I radiolabeling and for use in imaging studies to quantify the brain penetration of BAF312.     

Heterologous Expression of an Unusual Ketosynthase,SxtA, Leads to Production of Saxitoxin Intermediates in Escherichia coli

Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity in vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity in vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-¹⁵N₂] arginine and [1,2-¹³C₂] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.     

Imaging of KCa3.1 Channels in Tumor Cells with PET and Small-Molecule Fluorescent Probes

The Ca²⁺ activated K⁺ channel K_Ca3.1 is overexpressed in several human tumor cell lines, e. g. clear cell renal carcinoma, prostate cancer, non-small cell lung cancer. Highly aggressive cancer cells use this ion channel for key processes of the metastatic cascade such as migration, extravasation and invasion. Therefore, small molecules, which are able to image this K_Ca3.1 channel in vitro and in vivo represent valuable diagnostic and prognostic tool compounds. The [¹⁸F]fluoroethyltriazolyl substituted senicapoc was used as positron emission tomography (PET) tracer and showed promising properties for imaging of K_Ca3.1 channels in lung adenocarcinoma cells in mice. The novel senicapoc BODIPY conjugates with two F-atoms ( 9 a ) and with a F-atom and a methoxy moiety ( 9 b ) at the B-atom led to the characteristic punctate staining pattern resulting from labeling of single K_Ca3.1 channels in A549-3R cells. This punctate pattern was completely removed by preincubation with an excess of senicapoc confirming the high specificity of K_Ca3.1 labeling. Due to the methoxy moiety at the B-atom and the additional oxyethylene unit in the spacer, 9 b exhibits higher polarity, which improves solubility and handling without reduction of fluorescence quantum yield. Docking studies using a cryo-electron microscopy (EM) structure of the K_Ca3.1 channel confirmed the interaction of 9 a and 9 b with a binding pocket in the channel pore.     

Nuclear magnetic resonance spectroscopy spectroscopic investigation of the aging mechanism of polyethylene terephthalate vascular prostheses

This article attempts to develop and prove a technique to determine the degradation of polyethylene terephthalate (PET) for vascular prostheses. The implicit goal is to be able to quantify the amount of degradation to study the effect of in vivo aging. Nuclear magnetic resonance spectroscopy (¹H‐NMR) provides a comprehensive view of chemical macromolecular structures. Examination of a series of PET vascular prostheses showed significant chemical differences between the virgin prostheses and the explants collected after aging, especially for diethylene glycol and cyclic oligomers groups. Aging was investigated in terms of chemical scission of ester and ether linkages caused by hydrolytic reaction during the in vivo stay. Besides, we extended this ¹H‐NMR technique to determine hydroxyl end‐group concentrations and therefore the average number of macromolecular weight. To validate ¹H‐NMR results, complementary techniques, the chemical titration method and the classical viscosimetric method, were used. The results showed an increase of hydroxyl end‐group concentration and a decrease in the macromolecular weight for the explants. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Radiolabelled Cyclic Bisarylmercury: High Chemical and in vivo Stability for Theranostics

We show the synthesis of an in vivo stable mercury compound with functionality suitable for radiopharmaceuticals. The designed cyclic bisarylmercury was based on the water tolerance of organomercurials, higher bond dissociation energy of Hg−Ph to Hg−S, and the experimental evidence that acyclic structures suffer significant cleavage of one of the Hg−R bonds. The bispidine motif was chosen for its in vivo stability, chemical accessibility, and functionalization properties. Radionuclide production results in ^197(m)HgCl₂(aq), so the desired mercury compound was formed via a water-tolerant organotin transmetallation. The Hg-bispidine compound showed high chemical stability in tests with an excess of sulfur-containing competitors and high in vivo stability, without any observable protein interaction by human serum assay, and good organ clearance demonstrated by biodistribution and SPECT studies in rats. In particular, no retention in the kidneys was observed, typical of unstable mercury compounds. The ^natHg analogue allowed full characterization by NMR and HRMS.     

In vivo studies of the biosynthesis of vernolic acid in the seed of Vernonia galamensis

In vivo radiotracer experiments using [1-¹⁴C]acetate as the precursor were conducted to investigate the biosynthesis of vernolic acid (12, 13-epoxy-cis-9-octadecenoic acid) in the seeds of Vernonia galamensis. The acetate precursor radioactively labeled vernolate in phosphatidylcholine (PC), diacylglycerol, and triacylglycerol. Time-course kinetics of the incorporation of the radioactive tracer indicated that vernolate is synthesized while the acyl moiety is esterified to PC. Pulsechase experiments provided additional supporting evidence that vernolate is synthesized while esterified to PC. These results are consistent with the hypothesis that linoleoyl PC is the precursor of vernoleoyl-PC. Subsequently, vernolate is quickly moved from the PC pool to the triacylglycerol pool, where it accumulates.     

Comparison of in Silico,Electrochemical, in Vitro and in Vivo Metabolism of a Homologous Series of (Radio)fluorinated σ1 Receptor Ligands Designed for Positron Emission Tomography

The imaging of σ₁ receptors in the brain by fluorinated radiotracers will be used for the validation of σ₁ receptors as drug targets as well as for differential diagnosis of diseases in the central nervous system. The biotransformation of four homologous fluorinated PET tracers 1′‐benzyl‐3‐(ω‐fluoromethyl to ω‐fluorobutyl)‐3H‐spiro[2]benzofuran‐1,4′‐piperidine] ([¹⁸F] 1 – 4 ) was investigated. In silico studies using fast metabolizer (FAME) software, electrochemical oxidations, in vitro studies with rat liver microsomes, and in vivo metabolism studies after application of the PET tracers [¹⁸F] 1 – 4 to mice were performed. Combined liquid chromatography and mass spectrometry (HPLC–MS) analysis allowed structural identification of non‐radioactive metabolites. Radio‐HPLC and radio‐TLC provided information about the presence of unchanged parent radiotracers and their radiometabolites. Radiometabolites were not found in the brain after application of [¹⁸F] 2 – 4 , but liver, plasma, and urine samples contained several radiometabolites. Less than 2 % of the injected dose of [¹⁸F] 4 reached the brain, rendering [¹⁸F] 4 less appropriate as a PET tracer than [¹⁸F] 2 and [¹⁸F] 3 . Compounds [¹⁸F] 2 and [¹⁸F] 3 possess the most promising properties for imaging of σ₁ receptors in the brain. High σ₁ affinity (K_i=0.59 nm ), low lipophilicity (logD_7.4=2.57), high brain penetration (4.6 % of injected dose after 30 min), and the absence of radiometabolites in the brain favor the fluoroethyl derivative [¹⁸F] 2 slightly over the fluoropropyl derivative [¹⁸F] 3 for human use.     

A Pyropheophorbide Analogue Containing a Fused Methoxy Cyclohexenone Ring System Shows Promising Cancer-Imaging Ability

Herein we report the synthesis, photophysical properties, positron emission tomography (PET) imaging and photodynamic therapy (PDT) efficacy of methyl 3-(1′-m-iodobenzyloxy)ethyl-3-devinyl-verdin 4 (with or without the ¹²⁴I isotope). The PET imaging ability and ex vivo biodistribution of [¹²⁴I] 4 were compared with the well-studied methyl [3-(¹²⁴1′-m-iodobenzyloxy)ethyl]-3-devinyl-pyropheophorbide-a methyl ester (PET-ONCO or [¹²⁴I] 2 ) and [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) in BALB/c mice bearing colon-26 tumors. Whole-body PET images of [¹²⁴I] 4 containing a fused methoxy cyclohexenone ring system showed excellent tumor contrast with time (72>48>24 h post-injection). Ex vivo biodistribution results indicate that relative to the current clinical standard [¹⁸F]FDG and [¹²⁴I] 2 in 2 % ethanol formulation, [¹²⁴I] 4 , at the same radioactive dose (25 μCi per mouse), showed higher tumor uptake at 24 h post-injection and longer tumor retention. In biological environments, compound 4 showed lower fluorescence and lower singlet oxygen yield than 2 , which is possibly due to higher aggregation caused by the presence of a fused cyclohexenone ring system, resulting in limited in vitro/in vivo PDT efficacy. Therefore, the chlorophyll-a analogue [¹²⁴I] 4 provides easy access to a novel PET imaging agent (with no skin phototoxicity) to image cancer types—brain, renal carcinomas, pancreas—in which [¹⁸F]FDG shows limitations.     

Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

This study constructed cell-free glycolytic enzyme systems and compared them to their in vivo functions in Escherichia coli. Under in vitro conditions, flux regulation followed enzyme concentrations and kinetics. In E. coli, only one of the isozymes of phosphofructokinase (PfkA) and fructose-bisphosphate aldolase (FbaA) facilitate Embden-Meyerhof-Parnas (EMP) flux, but under in vitro assays, these isozymes were interchangeable. Additionally, in vitro introduction of the Entner–Doudoroff (ED) pathway improved glycolysis rates, while in vivo overexpression of the ED pathway could not capture significant flux unless its phosphotransferase system (PTS) was knocked out. Lastly, in vivo dynamic ¹³ C-experiments revealed that the labeling order of EMP pathway intermediates was not strictly cascade, indicating intracellular metabolites were not well mixed. These enigmatic observations cannot be fully explained by thermodynamics or substrate level regulations. This article supports the long-time conjecture that EMP enzymes are channeled, and the PTS may be an anchor point to initiate enzyme assemblies. © 2018 American Institute of Chemical Engineers AIChE J, 65: 483–490, 2019