The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

PSMA-targeted small-molecule drug-conjugates with valine-citrulline and phosphoramidate cleavable linkers.

In this study, we present a modular synthesis and evaluation of two prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugates (SMDCs) incorporating the potent chemotherapeutic agent monomethyl auristatin E (MMAE). These SMDCs are distinguished by their cleavable linker modules: one utilizing the widely known valine-citrulline linker, susceptible to cleavage by cathepsin B, and the other featuring a novel acid-labile phosphoramidate-based (PhosAm) linker. Both SMDCs maintained nanomolar affinity to PSMA. Furthermore, we confirmed the selective release of the payload and observed chemotherapeutic efficacy specifically within PSMA-positive prostate cancer cells, while maintaining cell viability in PSMA-negative cells. These findings not only validate the efficacy of our approach but also highlight the potential of the innovative pH-responsive PhosAm linker. This study contributes significantly to the field and also paves the way for future advancements in targeted cancer therapy.

PSMA-targeted SMART molecules outfitted with SN38.

Herein, we report the modular synthesis and evaluation of a prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugate (SMDC) carrying the chemotherapeutic agent, SN38. Due to the fluorogenic properties of SN38, payload release kinetics from the platform was observed in buffers representing the pH conditions of systemic circulation and cellular internalization. It was found that this platform is stable with minimal payload release at physiological pH with most rapid payload release observed at pH values representing the endosome complex. We confirmed selective payload release and chemotherapeutic efficacy for PSMA(+) prostate cancer cells over PSMA(-) cells. These results demonstrate that chemotherapeutic agents with limited solubility can be conjugated to a water-soluble targeting and linker platform without attenuating efficacy.

A PSMA-targeted doxorubicin small-molecule drug conjugate.

We developed a model small-molecule drug conjugate (SMDC) that employed doxorubicin as a representative chemotherapeutic targeted to the cell membrane biomarker PSMA (prostate-specific membrane antigen) expressed on prostate cancer cells. The strategy capitalized on the clatherin-mediated internalization of PSMA to facilitate the selective uptake and release of doxorubicin in the target cells. The SMDC was prepared and assessed for binding kinetics, plasma stability, cell toxicity, and specificity towards PSMA expressing prostate cancer cell lines. We observed high affinity of the SMDC for PSMA (IC50 5 nM) with irreversible binding, as well as specific effectiveness against PSMA(+) cells. These findings validated the strategy for a small molecule-based approach in targeted cancer therapy.

Structural, Biochemical, and Computational Characterization of Sulfamides as Bimetallic Peptidase Inhibitors.

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

Phosphorus containing analogues of SAHA as inhibitors of HDACs.

Histone deacetylases (HDACs) are a family of enzymes responsible for regulating DNA transcription by modulating its binding to histone proteins. HDACs are overexpressed in several types of cancers and are recognised as drug targets. Vorinostat, or suberanilohydroxamic acid (SAHA), is an histone deacetylase (HDAC) inhibitor with a hydroxamic acid as a zinc-binding group (ZBG), and it has been FDA approved for the treatment of T-cell lymphoma. In this work, phosphorus-based SAHA analogues were synthesised to assess their zinc-binding effectiveness compared to the hydroxamic acid of SAHA. Specifically, we examined phosphate, phosphoramidate and phosphorothiolate groups as isosteres of the canonical hydroxamic acid motif of conventional HDAC inhibitors. The compounds were screened for binding to HDAC enzymes from HeLa cell lysate. The most potent derivatives were then screened against HDAC3 and HDAC8 isoforms. HDAC inhibition assays demonstrated that these phosphorus-based SAHA analogs exhibited slow binding to HDACs but with greater potency than phosphonate SAHA analogs examined previously. All compounds inhibited HDACs, the most potent having an IC50 of 50 µM.

Prostate-Specific Membrane Antigen-Targeted Turn-on Probe for Imaging Cargo Release in Prostate Cancer Cells.

The tunable nature of phosphoramidate linkers enables broad applicability as pH-triggered controlled-release platforms, particularly in the context of antibody- and small-molecule-drug conjugates (ADCs and SMDCs), where there remains a need for new linker technology. Herein, we explored in-depth the release of turn-on fluorogenic payloads from a homoserinyl-based phosphoramidate acid-cleavable linker. Kinetics of payload release from the scaffold was observed in buffers representing the pH conditions of systemic circulation, early and late endosomes, and lysosomes. It was found that payload release takes place in two key consecutive steps: (1) P-N bond hydrolysis and (2) spacer immolation. These two steps were found to follow pseudo-first-order kinetics and had opposite dependencies on pH. P-N bond hydrolysis increased with decreasing pH, while spacer immolation was most rapid at physiological pH. Despite the contrasting release kinetics of these two steps, maximal payload release was observed at the mildly acidic pH (5.0-5.5), while minimal payload release occurred at physiological pH. We integrated this phosphoramidate-payload linker system into a PSMA-targeted fluorescent turn-on probe to study the intracellular trafficking and release of a fluorescent payload in PSMA-expressing prostate cancer cells. Results showed excellent turn-on and accumulation of the coumarin payload in the late endosomal and lysosomal compartments of these cells. The release properties of this linker mark it as an attractive alternative in the modular design of ADCs and SMDCs, which demand selective intracellular payload release triggered by the pH changes that accompany intracellular trafficking.

Corrigendum to "A click-ready pH-triggered phosphoramidate-based linker for controlled release of monomethyl auristatin E" [Tetrahedron Lett. Volume 61, Issue 41, 8 October 2020, 152398].

[This corrects the article PMC7665082.].

Inhibition of Acetylcholinesterases by Stereoisomeric Organophosphorus Compounds Containing Both Thioester and p-Nitrophenyl Leaving Groups.

Studies with acetylcholinesterase (AChE) inhibited by organophosphorus (OP) compounds with two chiral centers can serve as models or surrogates for understanding the rate, orientation, and postinhibitory mechanisms by the nerve agent soman that possesses dual phosphorus and carbon chiral centers. In the current approach, stereoisomers of O-methyl, [S-(succinic acid, diethyl ester), O-(4-nitrophenyl) phosphorothiolate (MSNPs) were synthesized, and the inhibition, reactivation, and aging mechanisms were studied with electric eel AChE (eeAChE) and recombinant mouse brain AChE (rmAChE). The MSNP RPRC isomer was the strongest inhibitor of both eeAChE and rmAChE at 8- and 24-fold greater potency, respectively, than the weakest SPSC isomer. eeAChE inhibited by the RPRC- or RPSC-MSNP isomer underwent spontaneous reactivation ∼10- to 20-fold faster than the enzyme inhibited by SPRC- and SPSC-MSNP, and only 4% spontaneous reactivation was observed from the SPRC-eeAChE adduct. Using 2-pyridine aldoxime methiodide (2-PAM) or trimedoxime (TMB-4), eeAChE inhibited by RPRC- or SPRC-MSNP reactivated up to 90% and 3- to 4-fold faster than eeAChE inhibited by the RPSC- or SPSC-MSNP isomer. Spontaneous reactivation rates for rmAChE were 1.5- to 10-fold higher following inhibition by RPSC- and SPSC-MSNPs than inhibition by either RC isomer, a trend opposite to that found for eeAChE. Oxime reactivation of rmAChE following inhibition by RPRC- and SPRC-MSNPs was 2.5- to 5-fold faster than inhibition by RPSC- or SPSC-MSNPs. Due to structural similarities, MSNPs that phosphylate AChE with the loss of the p-nitrophenoxy (PNP) group form identical, nonreactivatable adducts to those formed from SP-isomalathion; however, all the MSNP isomers inhibited AChE to form adducts that reactivated. Thus, MSNPs inactivate AChE via the ejection of either PNP or thiosuccinyl groups to form a combination of reactivatable and nonreactivatable adducts, and this differs from the mechanism of AChE inhibition by isomalathion.

A click-ready pH-triggered phosphoramidate-based linker for controlled release of monomethyl auristatin E.

In this work, we developed a novel "click"-ready pH-cleavable phosphoramidate linker for controlled-release of monomethyl auristantin E (MMAE) in antibody- and small molecule-drug conjugates application. This water-soluble linker was found to have tremendous stability at physiological pHs while rapidly releasing its payload at acidic pH. The linker can also be tailored to release payloads of diverse functional groups, broadening its applications.

Tunable pH-Sensitive 2-Carboxybenzyl Phosphoramidate Cleavable Linkers.

We previously described a pH-sensitive phosphoramidate linker scaffold that can be tuned to release amine-containing drugs at various pH values. In these previous studies it was determined that the tunability of this linker was dependent upon the proximity of an acidic group (e.g., carboxylic acid or pyridinium). In this study, we confirmed that the tunability of pH-triggered amine-release was also dependent upon the pKa of the proximal acidic group. A series of 2-carboxybenzyl phosphoramidates was prepared in which the pKa of the proximal benzoic acid was predictably attenuated by substituents on the benzoate ring consistent with their σ-values.

Phosphoramidate derivates as controlled-release prodrugs of l-Dopa.

l-Dopa has continued to be a mainstay in the symptomatic treatment of Parkinson's disease (PD). However, extensive peripheral metabolism, a short systemic circulation half-life and development of motor complications called dyskinesia prevents its long-term utilization as a PD therapeutic. Herein, we report a series of phosphoramidate derivatives of l-Dopa and controlled release of l-Dopa at pH 7.4 and 3. The kinetic data for the release of l-Dopa support our hypothesis that a proximal carboxylic acid can promote the pH-triggered hydrolysis of the phosphoramidate PN bond. As expected, esterification of the proximal carboxylic acid protects the scaffold from rapid release at low pH. This latter observation is particularly noteworthy as it suggests that the phosphoramidate-based l-Dopa-conjugate scaffold can be adapted for oral administration as an ester prodrug.

Bis(benzoyl) phosphate inactivators of beta-lactamase C from Mtb.

The class A ß-lactamase BlaC is a cell surface expressed serine hydrolase of Mycobacterium tuberculosis (Mtb), one of the causative agents for Tuberculosis in humans. Mtb has demonstrated increased susceptibility to ß-lactam antibiotics upon inactivation of BlaC; thus, making BlaC a rational enzyme target for therapeutic agents. Herein, we present the synthesis and structure-activity-relationship data for the 1st-generation library of bis(benzoyl) phosphates (1-10). Substituent effects ranged from σp = -0.27 to 0.78 for electronic and π = -0.41 to 1.98 for hydrophobic parameters. Compounds 1, 4 and 5 demonstrated the greatest inhibitory potency against BlaC in a time-dependent manner (kobs = 0.212, 0.324, and 0.450 mn-1 respectively). Combined crystal structure data and mass spectrometric analysis of a tryptic digest for BlaC inactivated with 4 provided evidence that the mechanism of inactivation by this bis(benzoyl) phosphate scaffold occurs via phosphorylation of the active-site Ser-70, ultimately leading to an aged form of the enzyme.

Crystal Structure of Phosphoserine BlaC from Mycobacterium tuberculosis Inactivated by Bis(Benzoyl) Phosphate.

Mycobacterium tuberculosis, the pathogen responsible for tuberculosis (TB), is the leading cause of death from infectious disease worldwide. The class A serine ß-lactamase BlaC confers Mycobacterium tuberculosis resistance to conventional ß-lactam antibiotics. As the primary mechanism of bacterial resistance to ß-lactam antibiotics, the expression of a ß-lactamase by Mycobacterium tuberculosis results in hydrolysis of the ß-lactam ring and deactivation of these antibiotics. In this study, we conducted protein X-ray crystallographic analysis of the inactivation of BlaC, upon exposure to the inhibitor bis(benzoyl) phosphate. Crystal structure data confirms that serine ß-lactamase is phosphorylated at the catalytic serine residue (Ser-70) by this phosphate-based inactivator. This new crystallographic evidence suggests a mechanism for phosphorylation of BlaC inhibition by bis(benzoyl) phosphate over acylation. Additionally, we confirmed that bis(benzoyl) phosphate inactivated BlaC in a time-dependent manner.

Tunable pH-Sensitive Linker for Controlled Release.

We have developed a novel pH-sensitive linker based on a phosphoramidate scaffold that can be tuned to release amine-containing drug molecules at various pH values. The pH-triggered phosphoramidate-based linkers are responsive to pH alone and do not require intracellular enzymatic action to initiate drug release. Key to the pH-triggered amine release from these linkers is a proximal acidic group (e.g., pyridinium or carboxylic acid) to promote the hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general-acid type mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine (e.g., primary, secondary, aniline). However, the proximity of the neighboring pyridine group attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. Based on the model scaffolds examined, phosphoramidate-based linkers could be selected for particular properties for controlled-release applications such as amine type, stability under physiological conditions, or release rates at various pH values such as intracellular endosomal conditions. The tunability of the phosphoramidate scaffold is expected to find broad applicability in various controlled drug-release applications such as antibody or small-molecule drug conjugates, drug-eluting stents, prodrug activation, as well as intracellular trafficking studies in which pH changes can trigger the release of turn-on dyes.

Second-Generation Tunable pH-Sensitive Phosphoramidate-Based Linkers for Controlled Release.

We developed a second generation of tunable pH-sensitive linkers based on our phosphoramidate scaffold to release amine-containing drugs under acidic conditions. The pH-triggered phosphoramidate-based linkers are responsive to pH and do not require intracellular enzymatic action to initiate drug release. On the basis of the model scaffolds examined, phosphoramidate-based linkers were selected for particular properties for controlled release applications such as amine type, stability under physiological conditions, or release rates at various pH values such as intracellular endosomal conditions. Key to the pH-triggered amine release from these linker is a proximal carboxylic acid to promote hydrolysis of the phosphoramidate P-N bond, presumably through an intramolecular general acid-type mechanism. Phosphoramidate hydrolysis is largely governed by the pKa of the leaving amine. However, the proximity of the neighboring carboxylic acid attenuates the stability of the P-N bond to hydrolysis, thus allowing for control over the release of an amine from the phosphoramidate center. In addition, we observed that the Thorpe-Ingold effect and rigidification of the scaffold could further enhance the rate of release. Esterification of the neighboring carboxylic acid was found to protect the scaffold from rapid release at low pH. This latter observation is particularly noteworthy as it suggests that the phosphoramidate-based drug-conjugate scaffold can be protected as an ester prodrug for oral administration. While the tunability phosphoramidate linkers is attractive for applications in intracellular trafficking studies in which pH changes can trigger release of turn-on dyes, antibody drug conjugates, small-molecule drug conjugates, and drug eluting stents (DES), the promise of oral delivery of drug conjugates is expected to have broad impact in controlled release applications.

A method to determine the mode of binding for GCPII inhibitors using bio-layer interferometry.

The rapid dilution of the enzyme-inhibitor complex assay to monitor the recovery of enzyme activity is a well-established assay to determine the reversibility of inhibition. Our laboratory has previously employed this method to ascertain the reversibility of known glutamate carboxypeptidase II (GCPII)-targeting agents. Due to the tedious and time-consuming nature of the assay, we sought to develop a facile method to determine the reversibility of well-characterized GCPII inhibitors using bio-layer interferometry (BLI). The results from the BLI assay are in agreement with the rapid dilution method. Herein, we report for the first time, a rapid, novel real-time BLI method to determine reversibility of inhibition.

Phosphoramidate-based peptidomimetic inhibitors of membrane type-1 matrix metalloproteinase.

Membrane-type I matrix metalloproteinases (MT1-MMP) is an enzyme critical to the remodeling and homeostasis of extracellular matrix, and when over expressed it contributes to metastasis and cancer cell progression. Because of its role and implication as a biomarker that is upregulated in various cancers, MT1-MMP has become an attractive target for drug discovery. A small pilot library of peptidomimetics containing a phosphoramidate core as a zinc-binding group was synthesized and tested for inhibitory potency against MT1-MMP. From this library, a novel two residue peptidomimetic scaffold was identified that confers potency against MT1-MMP at submicromolar concentrations. The results of this study confirm that for this scaffold, valine is favored as a P1 residue and leucine in the P1' position. Furthermore, steric tolerance was observed for the N-terminus, thus implicating that a second-generation library could be constructed to extend the scaffold to P2 without concomitant loss of affinity within the MT1-MMP catalytic domain.

Synthesis and evaluation of constrained phosphoramidate inhibitors of prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA) is a cell-surface enzyme-biomarker that is actively pursued for targeted delivery of imaging and therapeutic agents for prostate cancer. Our lab has developed PSMA inhibitors based on a phosphoramidate scaffold, which has shown both high selectivity for PSMA-positive tumors and rapid clearance in vivo when radiolabeled with (18)F. However, this scaffold exhibits hydrolytic instability under low pH and high temperature conditions, barring the use of other imaging or therapeutic radionuclides such as (68)Ga or (177)Lu. Previous studies in our lab have shown a trend in increasing acid stability as the distance between the phosphoramidate core and the α-carboxylate of the P1 residue is increased. Therefore, a new generation of phosphoramidate inhibitors was developed based on trans-4-hydroxyproline as the P1 residue to restrict the interaction of the α-carboxylate to the phosphoramidate core. These hydroxyproline inhibitors demonstrated comparable IC50 values to earlier generations as well as enhanced thermal and acid stability.

Biochemical characterization of prostate-specific membrane antigen from canine prostate carcinoma cells.

BACKGROUND: Prostate-specific membrane antigen (PSMA) remains an important target for diagnostic and therapeutic application for human prostate cancer. Model cell lines have been recently developed to study canine prostate cancer but their PSMA expression and enzymatic activity have not been elucidated. The present study was focused on determining PSMA expression in these model canine cell lines and the use of fluorescent small-molecule enzyme inhibitors to detect canine PSMA expression by flow cytometry. METHODS: Western blot and RT-PCR were used to determine the transcriptional and translational expression of PSMA on the canine cell lines Leo and Ace-1. An endpoint HPLC-based assay was used to monitor the enzymatic activity of canine PSMA and the potency of enzyme inhibitors. Flow cytometry was used to detect the PSMA expressed on Leo and Ace-1 cells using a fluorescently tagged PSMA enzyme inhibitor. RESULTS: Canine PSMA expression on the Leo cell line was confirmed by Western blot and RT-PCR, the enzyme activity, and flow cytometry. Kinetic parameters Km and Vmax of PSMA enzymatic activity for the synthetic substrate (PABGγG) were determined to be 393 nM and 220 pmol min(-1) mg protein(-1) , respectively. The inhibitor core 1 and fluorescent inhibitor 2 were found to be potent reversible inhibitors (IC50 = 13.2 and 1.6 nM, respectively) of PSMA expressed on the Leo cell line. Fluorescent labeling of Leo cells demonstrated that the fluorescent PSMA inhibitor 2 can be used for the detection of PSMA-positive canine prostate tumor cells. Expression of PSMA on Ace-1 was low and not detectable by flow cytometry. CONCLUSIONS: The results described herein have demonstrated that PSMA is expressed on canine prostate tumor cells and exhibits similar enzymatic characteristics as human PSMA. The findings show that the small molecule enzyme inhibitors currently being studied for use in diagnosis and therapy of human prostate cancer can also be extended to include canine prostate cancer. Importantly, the findings demonstrate that the potential of the inhibitors for use in diagnosis and therapy can be evaluated in an immunocompetent animal model that naturally develops prostate cancer before use in humans.