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Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. The cleavage of full-length cyclin E (FL-cycE) to low-molecular weight isoforms (LMW-E) dramatically alters substrate specificity, promoting G1-S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. In this study, analysis of tumor samples of patients with TNBC revealed that coexpression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared with FL-cycE, LMW-E specifically upregulates PKMYT1 expression and protein stability, thereby increasing CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E-dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breakage, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and patient-derived xenografts of LMW-E-high TNBC but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E-high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype. Significance: PKMYT1 upregulation and CDK1 phosphorylation in triple-negative breast cancer expressing low-molecular weight cyclin E leads to suboptimal responses to chemotherapy but sensitizes tumors to PKMYT1 inhibitors, proposing a personalized treatment strategy., (©2024 American Association for Cancer Research.)

Endocrine therapies (ET) with cyclin-dependent kinase 4/6 (CDK4/6) inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of patient-derived xenografts (PDXs) from patients with 22 ER+ breast cancer demonstrated that protein kinase, membrane-associated tyrosine/threonine one (PKMYT1), a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX-derived organoids and PDXs, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer., (©2024 The Authors; Published by the American Association for Cancer Research.)

Ovarian cancers (OVCAs) and endometrial cancers (EMCAs) with CCNE1- amplification are often resistant to standard of care treatment and represent an unmet clinical need. Previously, synthetic-lethal screening identified loss of the CDK1 regulator, PKMYT1, as synthetically lethal with CCNE1 -amplification. We hypothesized that CCNE1 -amplification associated replication stress will be more effectively targeted by combining the PKMYT1 inhibitor, lunresertib (RP-6306), with the ATR inhibitor, camonsertib (RP-3500/RG6526). Low dose combination RP-6306 with RP-3500 synergistically increased cytotoxicity more in CCNE1 amplified compared to non-amplified cells. Combination treatment produced durable antitumor activity and increased survival in CCNE1 amplified patient-derived and cell line-derived xenografts. Mechanistically, low doses of RP-6306 with RP-3500 increase CDK1 activation more so than monotherapy, triggering rapid and robust induction of premature mitosis, DNA damage and apoptosis in a CCNE1 -dependent manner. These findings suggest that targeting CDK1 activity by combining RP-6306 with RP-3500 is a novel therapeutic approach to treat CCNE1 -amplifed OVCAs and EMCAs., Competing Interests: Additional Declarations: Yes there is potential Competing Interest. F.S. serves on scientific advisory boards for AstraZeneca, GSK and Zentalis Pharmaceuticals. She has received institutional research funding from AstraZeneca, Repare Therapeutics, Instill Bio and Sierra Oncology.

PKMYT1 is a regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1. To address this need compound 1 was identified as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol increased potency on PKMYT1. These dimethylphenol analogs were found to exist as atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design enabled optimization of cell-based potency. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1. RP-6306 inhibits CCNE1 -amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials for treatment of various solid tumors.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies 1-4 . To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers., (© 2022. The Author(s).)

Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBCs) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces sickle hemoglobin polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in nonhuman primates (NHPs) and in healthy human subjects and evaluated the effects in RBCs from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHPs and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. After daily dosing of etavopivat over 5 consecutive days in NHPs, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBCs collected from patients with SCD with either homozygous hemoglobin S or hemoglobin S and C disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. SIGNIFICANCE STATEMENT: Etavopivat, a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase, decreased 2,3-diphosphoglycerate in red blood cells (RBCs) from nonhuman primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBCs from donors with sickle cell disease (SCD) (homozygous hemoglobin S or hemoglobin S and C genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD that could potentially reduce vaso-occlusion and improve anemia., (Copyright © 2022 The Author(s).)

Identification of low-dose, low-molecular-weight, drug-like inhibitors of protein-protein interactions (PPIs) is a challenging area of research. Despite the challenges, the therapeutic potential of PPI inhibition has driven significant efforts toward this goal. Adding to recent success in this area, we describe herein our efforts to optimize a novel purine carboxylic acid-derived inhibitor of the HDM2-p53 PPI into a series of low-projected dose inhibitors with overall favorable pharmacokinetic and physical properties. Ultimately, a strategy focused on leveraging known binding hot spots coupled with biostructural information to guide the design of conformationally constrained analogs and a focus on efficiency metrics led to the discovery of MK-4688 (compound 56 ), a highly potent, selective, and low-molecular-weight inhibitor suitable for clinical investigation.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

N-Hydroxy-2-arylisoindoline-4-carboxamides are potent and selective inhibitors of HDAC11. The discovery, synthesis, and structure activity relationships of this novel series of inhibitors are reported. An advanced analog (FT895) displays promising cellular activity and pharmacokinetic properties that make it a useful tool to study the biology of HDAC11 and its potential use as a therapeutic target for oncology and inflammation indications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.

A series of carboxamide-substituted thiophenes demonstrating inhibition of JAK2 is described. Development of this chemical series began with the bioisosteric replacement of a urea substituent by a pyridyl ring. Issues of chemical and metabolic stability were solved using the results of both in vitro and in vivo studies, ultimately delivering compounds such as 24 and 25 that performed well in an acute PK/PD model measuring p-STAT5 inhibition., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This communication discusses the discovery of novel reverse tricyclic pyridones as inhibitors of Janus kinase 2 (JAK2). By using a kinase cross screening approach coupled with molecular modeling, a unique inhibitor-water interaction was discovered to impart excellent broad kinase selectivity. Improvements in intrinsic potency were achieved by utilizing a rapid library approach, while targeted structural changes to lower lipophilicity led to improved rat pharmacokinetics. This multi-pronged approach led to the identification of 31, which demonstrated encouraging rat pharmacokinetics, in vivo potency, and excellent off-target kinase selectivity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients.

The JAK-STAT pathway mediates signaling by cytokines, which control survival, proliferation, and differentiation of a variety of cells. In recent years, a single point mutation (V617F) in the tyrosine kinase JAK2 was found to be present with a high incidence in myeloproliferative disorders (MPDs). This mutation led to hyperactivation of JAK2, cytokine-independent signaling, and subsequent activation of downstream signaling networks. The genetic, biological, and physiological evidence suggests that JAK2 inhibitors could be effective in treating MPDs. De novo design efforts of new scaffolds identified 1-amino-5H-pyrido[4,3-b]indol-4-carboxamides as a new viable lead series. Subsequent optimization of cell potency, metabolic stability, and off-target activities of the leads led to the discovery of 7-(2-aminopyrimidin-5-yl)-1-{[(1R)-1-cyclopropyl-2,2,2-trifluoroethyl]amino}-5H-pyrido[4,3-b]indole-4-carboxamide (65). Compound 65 is a potent, orally active inhibitor of JAK2 with excellent selectivity, PK profile, and in vivo efficacy in animal models.

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

This paper describes the discovery and design of a novel class of JAK2 inhibitors. Furthermore, we detail the optimization of a screening hit using ligand binding efficiency and log D. These efforts led to the identification of compound 41, which demonstrates in vivo activity in our study., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Polycythemia vera (PV) is a myeloproliferative disorder involving hematopoietic stem cells. A recurrent somatic missense mutation in JAK2 (JAK2V617F) is thought to play a causal role in PV. Therefore, targeting Jak2 will likely provide a molecular mechanism-based therapy for PV. To facilitate the development of such new and specific therapeutics, a suitable and well-characterized preclinical animal model is essential. Although several mouse models of PV have been reported, the spatiotemporal kinetics of PV formation and progression has not been studied. To address this, we created a bone marrow transplant mouse model that co-expresses mutant Jak2 and luciferase 2 (Luc2) genes. Bioluminescent imaging (BLI) was used to visualize disease cells and analyze the kinetics of PV development in vivo. To better understand the molecular mechanism of PV, we generated mice carrying a kinase inactive mutant Jak2 (Jak2K882E), demonstrating that the PV disease was dependent on constitutive activation of the Jak2 kinase activity. We further showed that the Jak2V617F mutation caused increased stem cell renewal activity and impaired cell differentiation, which was at least in part due to deregulated transcriptional programming. The Jak2V617F-Luc2 PV mice will be a useful preclinical model to characterize novel JAK2 inhibitors for the treatment of PV.

A number of inhibitors of kinesin spindle protein (KSP) have been described, which are known from X-ray crystallography studies to bind to an induced fit pocket defined by the L5 loop. We describe the characterization of eight mutant forms of KSP in which six residues that line this pocket have been altered. Mutants were analyzed by measuring rates of enzyme catalysis, in the presence and absence of six KSP inhibitors of four diverse structural classes and of varied ATP-competition status. Our analysis was in agreement with the model of binding established by the structural studies and suggests that binding energy is well distributed across functional groups in these molecules. The majority of the mutants retained significant enzymatic activity while diminishing inhibitor binding, indicating potential for the development of drug resistance. These data provide detailed information on interactions between inhibitor and binding pocket at the functional group level and enable the development of novel KSP inhibitors.

KIF14 is a microtubule motor protein whose elevated expression is associated with poor-prognosis breast cancer. Here we demonstrate KIF14 accumulation in mitotic cells, where it associated with developing spindle poles and spindle microtubules. Cells at later stages of mitosis were characterized by the concentration of KIF14 at the midbody. Time-lapse microscopy revealed that strong RNA interference (RNAi)-mediated silencing of KIF14 induced cytokinesis failure, causing several rounds of endoreduplication and resulting in multinucleated cells. Additionally, less efficacious KIF14-specific short interfering RNAs (siRNAs) induced multiple phenotypes, all of which resulted in acute apoptosis. Our data demonstrate the ability of siRNA-mediated silencing to generate epiallelic hypomorphs associated with KIF14 depletion. Furthermore, the link we observed between siRNA efficacy and phenotypic outcome indicates that distinct stages during cell cycle progression are disrupted by the differential modulation of KIF14 expression.

Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis. Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains. While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein. When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range. Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB. Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants. Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.

Nonribosomal peptide synthetases (NRPSs) are large, multidomain enzymes that biosynthesize medically important natural products. We report the crystal structure of the free-standing NRPS condensation (C) domain VibH, which catalyzes amide bond formation in the synthesis of vibriobactin, a Vibrio cholerae siderophore. Despite low sequence identity, NRPS condensation enzymes are structurally related to chloramphenicol acetyltransferase (CAT) and dihydrolipoamide acyltransferases. However, although the latter enzymes are homotrimers, VibH is a monomeric pseudodimer. The VibH structure is representative of both NRPS condensation and epimerization domains, as well as the condensation-variant cyclization domains, which are all expected to be monomers. Surprisingly, despite favorable positioning in the active site, a universally conserved histidine important in CAT and in other C domains is not critical for general base catalysis in VibH.

The glycopeptide antibiotics vancomycin and teicoplanin are vital components of modern anti-infective chemotherapy exhibiting outstanding activity against Gram-positive pathogens including members of the genera Streptococcus, Staphylococcus, and Enterococcus. These antibiotics also provide fascinating examples of the chemical and associated biosynthetic complexity exploitable in the synthesis of natural products by actinomycetes group of bacteria. We report the sequencing and annotation of the biosynthetic gene cluster for the glycopeptide antibiotic from Streptomyces toyocaensis NRRL15009, the first complete sequence for a teicoplanin class glycopeptide. The cluster includes 34 ORFs encompassing 68 kb and includes all of the genes predicted to be required to synthesize and regulate its biosynthesis. The gene cluster also contains ORFs encoding enzymes responsible for glycopeptide resistance. This role was confirmed by insertional inactivation of the d-Ala-d-lactate ligase, vanAst, which resulted in the predicted -sensitive phenotype and impaired antibiotic biosynthesis. These results provide increased understanding of the biosynthesis of these complex natural products.

The iron-chelating catechol siderophore vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit, ten-domain nonribosomal peptide synthetase system, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two (dihydroxyphenyl)methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have utilized site-specific and domain-deletion mutagenesis to map the heterocyclization and primary and secondary amine acylation activities of the six-domain (Cy1-Cy2-A-C1-PCP-C2) VibF subunit. We have found that Cy2 is capable of and limited to the condensation (amide bond formation) step of the three-step heterocyclization process, while Cy1 is capable of and limited to the final processing (cyclization/dehydration) steps to the completed heterocycle. Additionally, we have observed that the C2 domain functions in both N(9) (primary amine) acylation and N(5) (secondary amine) acylation of the (dihydroxybenzoyl)norspermidine substrate, leaving no catalytic role for the C1 domain, a conclusion confirmed with the formation of vibriobactin in a C1-deficient system. Thus VibF is an NRPS with two domains, Cy1 and Cy2, that perform a function otherwise performed by one and with one domain, C2, that performs a function otherwise performed by two. While C2 appeared to tolerate uncyclized threonine in place of the usual heterocycle in primary amine acylation, it refused this replacement in the corresponding donor substrate in secondary amine acylation.

Nonribosomal peptide synthetases are large enzyme complexes that synthesize a variety of peptide natural products through a thiotemplated mechanism. Assembly of the peptides proceeds through amino acid loading, amide-bond formation and chain translocation, and finally thioester lysis to release the product. The final products are often heavily modified, however, through methylation, epimerization, hydroxylation, heterocyclization, oxidative cross-linking and attachment of sugars. These activities are the province of specialized enzymes (either embedded in the multidomain nonribosomal peptide synthetase structure or standalone).

The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin. When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step. This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases. Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate. We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position.

Vibriobactin [N(1)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis[2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido]norspermidine] , is an iron chelator from the cholera-causing bacterium Vibrio cholerae. The six-domain, 270 kDa nonribosomal peptide synthetase (NRPS) VibF, a component of vibriobactin synthetase, has been heterologously expressed in Escherichia coli and purified. VibF has an unusual NRPS domain organization: cyclization-cyclization-adenylation-condensation-peptidyl carrier protein-condensation (Cy(1)-Cy(2)-A-C(1)-PCP-C(2)). VibF activates and covalently loads its PCP with L-threonine, and together with vibriobactin synthetase proteins VibE (adenylation) and VibB (aryl carrier protein) condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-VibF in the first demonstration of oxazoline formation by an NRPS cyclization domain. This enzyme-bound aryl oxazoline can be transferred by VibF to various amine acceptors but most efficiently to N(1)-(2, 3-dihydroxybenzoyl)norspermidine (k(cat) = 122 min(-1), K(m) = 1.7 microM), the product of 2,3-dihydroxybenzoyl-VibB, norspermidine, and VibH. This diacylated product undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed by VibF, to yield vibriobactin. Vibriobactin biosynthesis in vitro has thus been accomplished from four proteins, VibE, VibB, VibF, and VibH, with the substrates 2,3-dihydroxybenzoic acid, L-Thr, norspermidine, and ATP. Vibriobactin synthetase is an unusual NRPS in that all intermediates are not covalently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.

The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine. Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH. All three are homologous to nonribosomal peptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate ligase, VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), and VibH is a novel amide synthase that represents a free-standing condensation (C) domain. VibE and VibB are homologous to EntE and EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's ArCP domain. VibH then condenses this DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(1)-(2, 3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 min(-1) and a K(m) for acyl-VibB of 0.88 microM and for norspermidine of 1.5 mM. Exclusive monoacylation of a primary amine of norspermidine was observed. VibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, and hexylamine as substrates, albeit at lowered catalytic efficiencies. DHB-NSPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis. VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein-bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters.

The vancomycin resistance enzyme VanH is an alpha-ketoacid dehydrogenase that stereospecifically reduces pyruvate to D-lactate, which is required for the synthesis of the depsipeptide D-alanine-D-lactate. This compound then forms an integral part of the bacterial cell wall replacing the vancomycin target dipeptide D-alanine-D-alanine, thus the presence of VanH is essential for glycopeptide resistance. In this work, the VanH homologue from the glycopeptide antibiotic producing organism Streptomyces toyocaensis NRRL 15009, VanHst, has been overexpressed in Escherichia coli and purified, and its substrate specificity and mechanism were probed by steady-state kinetic methods and site-directed mutagenesis. The enzyme is highly efficient at pyruvate reduction with kcat/Km = 1.3 x 10(5) M-1 s-1 and has a more restricted alpha-ketoacid substrate specificity than VanH from vancomycin resistant enterococci (VRE). Conversely, VanHst shows no preference between NADH and NADPH while VanH from VRE prefers NADPH. The kinetic mechanism for VanHst was determined using product and dead-end inhibitors to be ordered BiBi with NADH binding first followed by pyruvate and products leaving in the order D-lactate, NAD+. Site-directed mutagenesis indicated that Arg237 plays a role in pyruvate binding and catalysis and that His298 is a candidate for an active-site proton donor. Glu266, which has been suggested to modulate the pKa of the catalytic His in other D-lactate dehydrogenases, was found to fulfill a similar role in VanHst, lowering a pKa value of kcat/Km nearly 2 units. These results now provide the framework for additional structure and inhibitor design work on the VanH family of antibiotic resistance enzymes.

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme in this process is a D-alanyl-D-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide D-alanyl-D-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for D-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.

The mechanism of high-level resistance to vancomycin in enterococci consists of the synthesis of peptidoglycan terminating in D-alanyl-D-lactate instead of the usual D-alanyl-D-alanine. This alternate cell wall biosynthesis pathway is ensured by the collective actions of three enzymes: VanH, VanA, and VanX. The origin of this resistance mechanism is unknown. We have cloned three genes encoding homologs of VanH, VanA, and VanX from two organisms which produce glycopeptide antibiotics: the A47934 producer Streptomyces toyocaensis NRRL 15009 and the vancomycin producer Amycolatopsis orientalis C329.2. The predicted amino acid sequences are highly similar to those found in VRE: 54 to 61% identity for VanH, 59 to 63% identity for VanA, and 61 to 64% identity for VanX. Furthermore, the orientations of the genes, vanH, vanA, and vanX, are identical to the orientations found in vancomycin-resistant enterococci. Southern analysis of total DNA from other glycopeptide-producing organisms, A. orientalis 18098 (chloro-eremomycin producer), A. orientalis subsp. lurida (ristocetin producer), and Amycolatopsis coloradensis subsp. labeda (teicoplanin and avoparcin producer), with a probe derived from the vanH, vanA, and vanX cluster from A. orientalis C329.2 revealed cross-hybridizing DNA in all strains. In addition, the vanH, vanA, vanX cluster was amplified from all glycopeptide-producing organisms by PCR with degenerate primers complementary to conserved regions in VanH and VanX. Thus, this gene sequence is common to all glycopeptide producers tested. These results suggest that glycopeptide-producing organisms may have been the source of resistance genes in vancomycin-resistant enterococci.

High level resistance to vancomycin and other glycopeptide antibiotics requires the synthesis of peptidoglycan terminating in the depsipeptide D-Ala-D-lactate, rather the usual D-Ala-D-Ala. We report the purification and enzymatic characterization of two D-Ala ligases from Streptomyces toyocaensis NRRL 15009 which produces the glycopeptide antibiotic A47934. One of these enzymes catalyzes only D-Ala-D-Ala peptide formation and is recovered from mid-exponential phase cell cultures. The other enzyme is a D-Ala-D-lactate ligase which can be detected in actively antibiotic producing stationary phase cultures or mid-exponential phase cultures grown in the presence of A47934. These results imply that peptidoglycan components of S. toyocaensis NRRL 15009 change upon induction of antibiotic production and predict the existence of a VanX-like D-Ala-D-Ala DD-dipeptidase activity.

The crisis in antibiotic resistance has resulted in an increasing fear of the emergence of untreatable organisms. Resistance to the glycopeptide antibiotic vancomycin in the enterococci, and the spread of these pathogens throughout the environment, has shown that this scenario is a matter of fact rather than fiction. The basis for vancomycin resistance is the manufacture of the depsipeptide D-Ala-D-lactate, which is incorporated into the peptidoglycan cell wall in place of the vancomycin target D-Ala-D-Ala. Pivotal to the resistance mechanism is the production of a D-Ala-D-Ala ligase capable of ester formation. Two highly efficient depsipeptide ligases have been cloned from vancomycin-resistant enterococci: VanA and VanB. These ligases show high amino acid sequence similarity to each other ( approximately 75%), but less so to other D-Ala-D-X ligases (<30%). We have cloned ddls from two glycopeptide-producing organisms, the vancomycin producer Amycolatopsis orientalis and the A47934 producer Streptomyces toyocaensis. These ligases show strong predicted amino acid homology to VanA and VanB (>60%) but not to other D-Ala-D-X ligases (<35%). The D-Ala-D-Ala ligase from S. toyocaensis shows D-Ala-D-lactate synthase activity in cell-free extracts of S. lividans transformed with the ddl gene and confirms the predicted enzymatic activity. These results imply a close evolutionary relationship between resistance mechanisms in the clinics and in drug-producing bacteria.

1. Single-ion-channel recording has been used to estimate the equilibrium concentration-response relationship for several acetylcholine analogues. The response, corrected for desensitization, was taken as the probability of a channel being open during clusters of openings that were separated by desensitized periods. 2. All agonists were able to block the channels which they themselves opened. Carbachol, suberyldicholine and the sulphonium analogue of acetylcholine were all found to be efficacious agonists in the sense that the results indicate that all of them, in sufficiently high concentration, would be able to open 90% or more of channels if it were not for channel block. 3. In the case of suberyldicholine the results are much as predicted by the interpretation of the fine structure of channel openings at low agonist concentrations. 4. The maximum probability of opening that could be obtained with decamethonium and with phenyltrimethylammonium was low (below 4%), and it was not possible to distinguish whether this was wholly a result of the powerful (relative to activation potency) channel-blocking action of these agonists, or whether it was to some extent attributable to their being genuine partial agonists. 5. The results suggest that, for a range of agonists, differences in equilibrium potency are usually more strongly influenced by affinity for binding to the resting state of the receptor than by ability to activate the receptor once bound, though in the case of suxamethonium (relative to acetylcholine) the contributions of each factor are similar.

1. Patch clamp methods were used to study the equilibrium and kinetic properties of the acetylcholine analogue, succinyldicholine (suxamethonium), which is used clinically as a neuromuscular blocking agent. 2. The equilibrium concentration-response curve, corrected for desensitization was estimated by measuring as the response the probability of being open of single ion channels during clusters of activity that occur between long desensitized periods. Suxamethonium (Sux) was about 7.6-fold less potent than acetylcholine (ACh) (at low concentrations), partly because of 2.9-fold lower affinity for the resting receptor, and partly because of a lower ability to activate the receptor once bound. 3. Sux was a more potent blocker of the open ion channel than ACh (equilibrium constant about 200 microM); this limited the maximum open probability to about 0.36 (at 12 degrees C and -120 mV). Individual channel blockages lasted about 65 microseconds on average. They appeared to get longer at high agonist concentration; however, a simulation method was used to show that this effect could be accounted for by the fact that at higher concentrations there are more openings that are too brief to be detected. Over the concentration range tested the effects were described by a simple open channel block mechanism. 4. No component of brief shut times could be detected other than those resulting from channel blockages. However, the results suggest that multiple channel openings (the nachschlag phenomenon) should be rare, so this is not inconsistent with previous results with other agonists. 5. Sux differed from ACh and carbachol in that it had a somewhat lower efficacy and a greater channel blocking action. However, in clinical practice channel block is unlikely to contribute to neuromuscular block to any significant extent; the main mechanism of paralysis, at least in the early stages, is probably a result of prolonged depolarization of the region of membrane surrounding the motor endplate leading to inactivation of the sodium channels therein.

1. Voltage-activated currents have been recorded from cerebellar granule neurones in explant cultures from young rats (1-9 days old). Cells were examined with whole-cell patch-clamp methods. Depolarizing pulses from a pre-pulse potential of -100 mV evoked a rapidly activated transient inward current, and an outward current which decayed in two phases. The ionic dependence, kinetics and pharmacological properties of these currents have been studied. 2. Peak inward Na+ currents in cells from 7-day-old rats were in the range 350-450 pA. No evidence was found for the presence of calcium currents. Thus, inward current was unchanged in zero Ca2+, 1 mM-EGTA solution. No inward current was obtained in medium containing 10 mM-Ba2+ and tetrodotoxin (TTX). Supplementing the pipette (i.e. intracellular) solution with Mg-ATP did not reveal any Ca2+ current. 3. Depolarizing steps (from -100 mV) in TTX-containing solution gave an early transient outward current and a late outward current. The transient current resembled IA described in other cells, and reversed close to EK in both normal and elevated potassium concentrations, indicating that K+ is the predominant charge carrier. Depolarizing steps from -50 mV failed to give a transient outward current, and gave only a slowly rising current which resembled the late potassium current, IK. 4. Inactivation of the transient current was examined by applying test depolarizations from increasingly negative pre-pulse potentials (-50 to -120 mV): half-inactivation occurred at -72 mV. Transient outward currents decayed exponentially with time constants, tau, of 7.3-25.3 ms at 0 mV. The time course of removal of inactivation in cells held at -50 mV, and given increasingly long pre-pulses to -100 mV, was exponential with tau = 35 ms. 5. Both transient and late outward currents were reversibly abolished by addition to the bathing medium of 10 mM-Ba2+ or 1 mM-quinine. Outward K+ current was not dependent on external calcium. Tetraethylammonium (20 mM) selectively reduced the late outward current; the peak transient current was reduced by less than 20%. 4-Aminopyridine (2 mM) showed little selectivity between transient and late outward currents. 6. It is concluded that cerebellar granule cells from young rats possess voltage-activated inward Na+ current as well as two types of K+ current, IA and IK. In terms of neuronal functioning, the properties of the transient outward current may confer a role in regulating excitability and in repolarization, but a definitive statement will require knowledge of the cellular location and relative densities of channels in granule cells in vivo.

Thousands of salivary cells fill the interstices throughout the anterior ends of jawed leeches. The somata are large (30-200 micron in diameter). They project single processes (ductules) into the three jaws, and were found to fire overshooting action potentials of 50-85 mV amplitude and 100-200 ms duration at low spontaneous rates. The action potentials were not detected in the presence of cobalt (10 mmol l-1), but could be recorded when sodium was absent from the Ringer, so they appear to be calcium-dependent. Salivary material is transported by the long processes of these unicellular glands and secreted into ducts which alternate with paired teeth on the jaws. Secretion is activated reliably by 10(-6) mol l-1 serotonin, but not by other neurotransmitters found in the leech nervous system. Each jaw secretes at an average rate of 230 nl min-1 in the presence of serotonin, and secretion is completely abolished by cobalt. Perfusion with serotonin excites the salivary gland cells into impulse activity, and often evokes bursting. Impulse activity of the peripherally projecting, serotonergic Retzius cells evokes both depolarizations and action potentials in the salivary gland cells. In jawed leeches, central neurones appear to control salivation by a peripheral release of serotonin. This neurotransmitter evokes calcium-dependent action potentials and calcium, in turn, stimulates secretion.

A quartzite believed to be at least 2700 million years old contains pockets of minute red or yellow objects presumably composed of hematite and goethite, respectively. The size and morphology of the objects suggests that most of them may be the remains of microorganisms of at least two species.