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Filamentous soil bacteria are known to produce diverse specialized metabolites. Despite having enormous potential as a source of pharmaceuticals, they often produce bioactive metabolites at low titers. Here, we show that inactivation of the pactamycin, NFAT-133, and conglobatin biosynthetic pathways in Streptomyces pactum ATCC 27456 significantly increases the production of the mitochondrial electron transport inhibitors piericidins. Similarly, inactivation of the pactamycin, NFAT-133, and piericidin pathways significantly increases the production of the heat-shock protein (Hsp) 90 inhibitor conglobatin. In addition, four new conglobatin analogues (B2, B3, F1, and F2) with altered polyketide backbones, together with the known analogue conglobatin B1, were identified in this mutant, indicating that the conglobatin biosynthetic machinery is promiscuous toward different substrates. Among the new conglobatin analogues, conglobatin F2 showed enhanced antitumor activity against HeLa and NCI-H460 cancer cell lines compared to conglobatin. Conglobatin F2 also inhibits colony formation of HeLa cells in a dose-dependent manner. Molecular modeling studies suggest that the new conglobatins bind to human Hsp90 and disrupt Hsp90/Cdc37 chaperone/co-chaperone interactions in the same manner as conglobatin. The study also showed that genes that are involved in piericidin biosynthesis are clustered in two different loci located distantly in the S. pactum genome.

Glycosylation is a common modification reaction in natural products biosynthesis and has been known to be a post assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation, and in vitro pathway reconstitution we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-[3-aminophenyl]3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free β-ketoacid that subsequently undergoes decarboxylation.

The first total synthesis of pactalactam was accomplished using substrate-controlled stereoselective aziridination and regioselective aziridine ring-opening to construct three continuous amino groups on an octasubstituted cyclopentane core. The cyclopentane framework was obtained by ring-closing metathesis and aldol coupling using a l-threonine-derived oxazoline compound. Cyclic urea formation, m-acetylphenyl group introduction by Chan-Lam coupling, and primary alcohol-selective acylation yielded the reported pactalactam structure. The presence of pactalactam in the fermentation broth of pactamycin-producing bacteria was also confirmed.

A formal total synthesis of pactamycin (1) has been accomplished by face-selective and regioselective nitroso Diels–Alder (NDA) reaction of acyl nitroso compound 14, which contains a camphorsultam ...

The use of β-nitroenamines as a new class of acceptors in the enantioselective Pd-catalyzed trimethylenemethane cycloaddition afforded differentiated 1,2-dinitrogen bearing cyclopentanes with three contiguous stereocenters. The utility of these acceptors was demonstrated with the efficient construction of the core of jogyamycin and aminocyclopentitols. Further elaboration of the cycloadducts provided a concise synthetic approach toward joygamycin.

Pactamycin is a structurally unique aminocyclitol antibiotic with broad-spectrum cell growth inhibitory activity. To explore the bountiful activity of the aminocyclitol core of pactamycin, an efficient, modular, and asymmetric synthesis of aminocyclopentitols resembling the pactamycin pharmacophore has been developed employing a SmI2-mediated imino-pinacol coupling strategy. Two of the compounds exhibited antitumor activity against A375 melanoma cells.

International audience; Dirhodium(II)-catalyzed nitrene transfers have been used to prepare a novel synthetic platform bearing the triamino moiety present in pactamycin, jogyamycin and cranomycin. Catalytic intramolecular C(sp3)–H amination and alkene aziridination reactions, the latter followed by a nucleophilic aziridine ring opening, are the key steps of this strategy.

The unique five-membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d-glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar-derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD+ -dependent alcohol dehydrogenase PctP and pyridoxal 5'-phosphate dependent aminotransferase PctC converted N-acetyl-d-glucosaminyl-3-aminoacetophonone into 3'-amino-3'-deoxy-N-acetyl-d-glucosaminyl-3-aminoacetophenone. Further, N-acetyl-d-glucosaminyl-3-aminophenyl-β-oxopropanoic acid ethyl ester was converted into the corresponding 3'-amino derivative. However, PctP did not oxidize most of the tested d-glucose derivatives, including UDP-GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.

Pactamycin, a potent antitumor and antimicrobial antibiotic, possesses a densely functionalized cyclopentane core structure. This paper describes the concise synthesis of an advanced intermediate for synthesizing the enantiomer of pactamycin that contains the cyclopentane skeleton bearing all the necessary amino functions with correct stereochemistries.

Jogyamycin is a member of the aminocyclopentitol class of natural products that exhibits significant antiprotozoal activities against diseases that include African sleeping sickness and malaria. Herein, we report a route to the core of this natural product via an underutilized Ichikawa rearrangement as a key step. This route efficiently forms the cyclopentane ring from simple and easily accessible starting materials, and rapidly installs the C1/C4/C5 polar functional groups. In addition, this strategy shows excellent potential for the preparation of analogues of jogyamycin to study how structural changes impact the selectivity in binding to the ribosome.

Molecules isolated from natural sources including bacteria, fungi, and plants are a long-standing source of therapeutics that continue to add to our medicinal arsenal today. Despite their potency and prominence in the clinic, complex natural products often exhibit a number of liabilities that hinder their development as therapeutics, which may be partially responsible for the current trend away from natural product discovery, research, and development. However, advances in synthetic biology and organic synthesis have inspired a new generation of natural product chemists to tackle powerful undeveloped scaffolds. In this Perspective, we will present case studies demonstrating the historical and current focus on making targeted, but significant, changes to natural product scaffolds via biosynthetic gene cluster manipulation, total synthesis, semisynthesis, or a combination of these methods, with a focus on increasing activity, decreasing toxicity, or improving chemical and pharmacological properties.

Pactamycin is an antibiotic produced by Streptomyces pactum with antitumor and antimalarial properties. Pactamycin has a unique aminocyclitol core that is decorated with 3-aminoacetophenone, 6-methylsaliciate, and an N,N-dimethylcarbamoyl group. Herein, we show that the adenylation enzyme PctU activates 3-aminobenzoic acid (3ABA) with adenosine triphosphate and ligates it to the holo form of the discrete acyl carrier protein PctK to yield 3ABA-PctK. Then, 3ABA-PctK is N-glycosylated with uridine diphosphate-N-acetyl-d-glucosamine (UDP-GlcNAc) by the glycosyltransferase PctL to yield GlcNAc-3ABA-PctK. Because 3ABA is known to be a precursor of the 3-aminoacetophenone moiety, PctU appears to be a gatekeeper that selects the appropriate 3-aminobenzoate starter unit. Overall, we propose that acyl carrier protein-bound glycosylated 3ABA derivatives are biosynthetic intermediates of pactamycin biosynthesis.

The antitumor antibiotic pactamycin is a highly substituted aminocyclopentitol-derived secondary metabolite produced by the soil bacterium Streptomyces pactum. It has exhibited potent antibacterial, antitumor, antiviral, and antiprotozoal activities. Despite its outstanding biological activities, the complex chemical structure and broad-spectrum toxicity have hampered its development as a therapeutic, limiting its contribution to biomedical science to a role as a molecular probe for ribosomal function. However, a detailed understanding of its biosynthesis and how the biosynthesis is regulated has made it possible to tactically design and produce new pactamycin analogues, some of which have shown improved pharmacological properties. This mini-review describes the biosynthesis, regulation, engineered production, and biological activities of pactamycin and its congeners. It also highlights the suitability of biosynthetic methods as a feasible approach to generate new analogues of complex natural products and underscores the importance of utilizing biosynthetic enzymes as tools for chemoenzymatic production of structurally diverse bioactive compounds.

Pactamycin is a bacteria-derived aminocyclitol antibiotic with a wide-range of biological activity. Its chemical structure and potent biological activities have made it an interesting lead compound for drug discovery and development. Despite its unusual chemical structure, many aspects of its formation in nature remain elusive. Using a combination of genetic inactivation and metabolic analysis, we investigated the tailoring processes of pactamycin biosynthesis in Streptomyces pactum. The results provide insights into the sequence of events during the tailoring steps of pactamycin biosynthesis and explain the unusual production of various pactamycin analogues by S. pactum mutants. We also identified two new pactamycin analogues that have better selectivity indexes than pactamycin against malarial parasites.

Covering: up to mid-2018 Pyridoxal 5'-phosphate (PLP) is a versatile organic cofactor used to catalyze diverse reactions on amino acid, oxoacid, and amine substrates. Here we review the reactions catalyzed by PLP-dependent enzymes, highlighting enzymes reported in the natural product biosynthetic literature. We describe enzymes that catalyze transaminations, Claisen-like condensations, and β- and γ-eliminations and substitutions, along with epimerizations, decarboxylations, and transaldolations. Finally, we describe a newly reported group of O2-, PLP-dependent enzymes. Altogether, natural product biosynthesis showcases the incredible versatility of PLP-dependent transformations for building chemical complexity.

Mutational analysis of the pyridoxal 5'-phosphate (PLP)-dependent enzyme PctV was carried out to elucidate the multi-step reaction mechanism for the formation of 3-aminobenzoate (3-ABA) from 3-dehydroshikimate (3-DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5'-phosphate (PMP) with 3-DSA. The chemical structure of this intermediate was supported by X-ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3-ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.

The cyclopentane core framework 23 of pactamycin (1) was synthesized in 14 steps from symmetric cyclohexadiene 11. Our synthetic strategy features Rh-mediated catalytic desymmetrization of 10 via aziridination and then regioselective ring-opening reaction of sulfonylaziridine 9 with NaN3, ring-contraction of cyclohexene 14 by ozonolysis followed by intramolecular aldol reaction, and stereoselective construction of the sequential tetrasubstituted carbons by dihydroxylation and methylation reaction. Stereospecific incorporation of amine on tetrasubstituted carbon was achieved by Curtius rearrangement and subsequent carbamide formation.

β-Ketoacyl-acyl carrier protein (β-Ketoacyl-ACP) synthase (KAS) III catalyzes the first step in fatty acid biosynthesis, involving a Claisen condensation of the acetyl-CoA starter unit with the first extender unit, malonyl-ACP, to form acetoacetyl-ACP. KAS III-like proteins have also been reported to catalyze acyltransferase reactions using coenzyme A esters or discrete ACP-bound substrates. Here, we report the in vivo and in vitro characterizations of a KAS III-like protein (PtmR), which directly transfers a 6-methylsalicylyl moiety from an iterative type I polyketide synthase to an aminocyclopentitol unit in pactamycin biosynthesis. PtmR is highly promiscuous, recognizing a wide array of S-acyl-N-acetylcysteamines as substrates to produce a suite of pactamycin derivatives with diverse alkyl and aromatic features. The results suggest that KAS III-like proteins may be used as versatile tools for modifications of complex natural products.

The aminocyclitol family is a relatively new class of natural products such as gentamicin, kanamycin, and streptomycin, which have been used clinically for decades as potent antimicrobial agents. These secondary metabolites are chiefly produced by microorganisms, especially Actinomycetes. Their chemical structures most commonly contain a C7N unit, 2-epi-5-epi-valiolone or 3-amino-5-hydroxybenzoic acid (3,5-AHBA) which are known to be responsible for their biological activities. In the course of current study, the biosynthesis of the C7N-containing metabolites, validamycin and acarbose, pactamycin, have been evaluated. We studied N-formamide salicylic acid (FSA) moiety which is a C7N unit synthesized from tryptophan by microorganisms. A strong antifungal agent antimycin, isolated from several Streptomyces sp., contains an FSA moiety, and constitutes a unique nine-membered dilactone ring with L-threonine, short-chain fatty acid, and an amide linkage connecting it to an FSA moiety. Also, an antitumor antibiotic asukamycin, produced by Streptomyces nodosus subsp. asukaensis ATCC 29757, consists of both 3,4-AHBA and C5N, cyclohexane ring linked to trans-triens. To improve the efficacy and reduce the toxicity of these metabolites, further structural modification is needed. Total chemical synthesis of these complex compounds is difficult. Therefore, alternative approaches are required, e.g., biosynthetic or genetic modification methods. This review presents the biosynthetic study on these compounds for creating new analogs using mutasyntheis.

A mutasynthetic strategy has been used to generate fluorinated TM-025 and TM-026, two biosynthetically engineered pactamycin analogues produced by a mutant strain of Streptomyces pactum. The compounds were produced by blocking the formation of the pactamycin biosynthetic precursor 3-aminobenzoic acid and performing precursor-directed structure modification. The fluorinated compounds maintain excellent activity and selectivity toward chloroquine-sensitive and multidrug-resistant strains of malarial parasites as the parent compounds. The results also provide insights into the biosynthesis of 3-aminobenzoic acid in S. pactum.

This article describes synthetic studies that culminated in the first total synthesis of pactamycin and pactamycate and, in parallel, the two known congeners, de-6-MSA-pactamycin and de-6-MSA-pactamycate, lacking the 6-methylsalicylyl moiety. Starting with L-threonine as a chiron, a series of stereocontrolled condensations led to a key cyclopentenone harboring a spirocyclic oxazoline. A series of systematic functionalizations led initially to the incorrect cyclopentanone epoxide, which was "inverted" under solvolytic conditions. Installation of the remaining groups and manipulation of the oxazoline eventually led to pactamycin, pactamycate, and their desalicylyl analogues.

Pactamycin is an aminocyclopentitol-derived natural product that has potent antibacterial and antitumor activities. Sequence analysis of an 86 kb continuous region of the chromosome from Streptomyces pactum ATCC 27456 revealed a gene cluster involved in the biosynthesis of pactamycin. Gene inactivation of the Fe-S radical SAM oxidoreductase (ptmC) and the glycosyltransferase (ptmJ), individually abrogated pactamycin biosynthesis; this confirmed the involvement of the ptm gene cluster in pactamycin biosynthesis. The polyketide synthase gene (ptmQ) was found to support 6-methylsalicylic acid (6-MSA) synthesis in a heterologous host, S. lividans T7. In vivo inactivation of ptmQ in S. pactum impaired pactamycin and pactamycate production but led to production of two new pactamycin analogues, de-6-MSA-pactamycin and de-6-MSA-pactamycate. The new compounds showed equivalent cytotoxic and antibacterial activities with the corresponding parent molecules and shed more light on the structure-activity relationship of pactamycin.

[structure: see text]. Pactamycin, one of the most complex and densely functionalized aminocyclitol antibiotics known, presents synthetic challenges that include reactivity and sterics, relative and absolute stereochemistry, and functional group compatibility and protection. An approach is reported that features four different types of (cyclopentane) face selective functionalization reactions and results in a polyfunctionalized and appropriately protected intermediate that incorporates all the core carbons and the oxygenated functionality of the target.

The synthesis and biological analysis of a number of novel congeners of the aminocyclopentitol pactamycin is described. Specific attention was paid to the preparation of derivatives at crucial synthetic branch points of the parent structure, and biological assays revealed a number of insights into the source of pactamycin’s biological activity. Additionally, the encapsulation of pactamycin and select derivatives into the PRINT© nanoparticle technology was investigated as a proof-of-concept, and evidence of bioactivity modulation through nanoparticle delivery is demonstrated. This work has provided heretofore unrealized access to a large number of novel compounds for further evaluation.

Store-operated Ca(2+) entry (SOCE) is activated in response to depletion of intracellular Ca(2+) from the endoplasmic reticulum (ER). A variety of agonists stimulate SOCE via IP(3)-dependent Ca(2+) depletion. SOCE is also activated by thapsigargin, an inhibitor of Ca(2+) reuptake into the ER that induces a net Ca(2+) loss from the ER by unmasking a Ca(2+) "leak" pathway. The molecular identity of this Ca(2+) leak channel and the physiological conditions under which such agonist-independent Ca(2+) depletion might occur remain poorly characterized. In this study, we report that inhibition of the initiation step of protein synthesis (with pactamycin) resulted in detectable Ca(2+) depletion in ER and activation of SOCE. This was completely prevented if the ribosome-nascent chain complexes were first stabilized with an irreversible inhibitor of translational elongation (emetine), suggesting that ER Ca(2+) depletion had occurred through open translocons at the ER. Notably, emetine pretreatment also attenuated thapsigargin-mediated Ca(2+) release and SOCE. Furthermore, both pactamycin and thapsigargin stimulated translocation of STIM1, a protein required for activation of SOCE, to the subplasma membrane region and activated the SOCE-associated current, I (SOC). In aggregate, these data reveal an agonist-independent mechanism for internal Ca(2+) store depletion and activation of SOCE. We suggest that the functional coupling between SOCE and protein synthesis is likely to be critical for maintaining [Ca(2+)](ER) within a range that is required to prevent ER stress during changes in cellular translational activity.

A tricyclic compound including a cyclopentane structure for pactamycin, an antitumor antibiotic, was constructed by Overman rearrangement and Pauson-Khand cyclization as key steps starting from diacetone-D-glucose.

An asymmetric total synthesis of the aminocyclopentitol pactamycin is described. The title compound is delivered in 15 steps from 2,4-pentanedione. Critical to this approach was the exploitation of a complex symmetry-breaking reduction strategy to assemble the C1, C2, and C7 relative stereochemistry within the first four steps of the synthesis. Multiple iterations of this reduction strategy are described, and a thorough analysis of stereochemical outcomes is detailed. In the final case, an asymmetric Mannich reaction was developed to install a protected amine directly at the C2 position. Symmetry-breaking reduction of this material gave way to a remarkable series of stereochemical outcomes leading to the title compound without recourse to nonstrategic downstream manipulations. This synthesis is immediately accommodating to the preparation of structural analogs.

We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.

Overexpression of the translation initiation factor eIF4E leads to cell transformation and occurs in a number of human cancers [1]. mRNA translation and cell growth can be regulated through the availability of eIF4E to form initiation complexes by binding to eIF4G. The availability of eIF4E is blocked through the binding of members of a family of eIF4E-binding proteins (4E-BPs) [2] [3]. Indeed, cell transformation caused by the overexpression of eIF4E can be reversed by the overexpression of 4E-BPs [4] [5] [6] [7] [8]. To study the role of eIF4E in cell transformation, we developed a series of peptides based on the conserved eIF4E-binding motifs in 4E-BPs and eIF4G [9] linked to the penetratin peptide-carrier sequence, which mediates the rapid transport of peptides across cell membranes. Surprisingly, introduction of these eIF4E-binding peptides into MRC5 cells led to rapid, dose-dependent cell death, with characteristics of apoptosis. Single alanine substitutions at key positions in the peptides impair their binding to eIF4E and markedly reduce their ability to induce apoptosis. A triple alanine substitution, which abolishes binding to eIF4E, renders the peptide unable to induce apoptosis. Our data provide strong evidence that the peptides induce apoptosis through binding to eIF4E. They do not induce apoptosis through inhibition of protein synthesis, as chemical inhibitors of translation did not induce apoptosis or affect peptide-induced cell death. Thus these new data indicate that eIF4E has a direct role in controlling cell survival that is not linked to its known role in mRNA translation.

Previous studies suggest that post-transcriptional events play an important role in estrogen-induced loss of estrogen receptor expression. The present study shows that treatment of MCF-7 cells with estradiol resulted in a six-fold decrease in estrogen receptor mRNA half-life from 4 h in control cells to 40 min in estradiol treated cells. To determine the role of protein synthesis in the regulation of estrogen receptor mRNA stability, several translational inhibitors were utilized. Pactamycin and puromycin, which prevent ribosome association with mRNA, inhibited the effect of estradiol on receptor mRNA stability, whereas cycloheximide, which has no effect on ribosome association with mRNA, had no effect on estradiol regulation of estrogen receptor mRNA stability. In control cells, the total cellular content of estrogen receptor mRNA was associated with high molecular weight polyribosomes. Treatment with estradiol resulted in a 70% decrease in estrogen receptor mRNA associated with polyribosomes but had no effect on the polyribosome distribution of estrogen receptor mRNA. In an in vitro degradation assay, polyribosomes isolated from estradiol-treated cells degraded ER mRNA faster than polyribosomes isolated from control cells. The nuclease activity associated with the polysome fraction appeared to be Mg2+ independent and inhibited by RNasin. Freeze-thawing and heating at 90 degrees C for 10 min resulted in the loss of nuclease activity. These studies suggest that an estrogen-regulated nuclease activity associated with ribosomes alters the stability of estrogen receptor mRNA.

Natural amino donation: A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature.

An asymmetric total synthesis of the aminocyclopentitol pactamycin is described, which delivers the title compound in 15 steps from 2,4-pentanedione. Critical to this approach was the exploitation of a complex symmetry-breaking reduction strategy to assemble the C1, C2, and C7 relative stereochemistry within the first four steps of the synthesis. Multiple iterations of this reduction strategy are described, and a thorough analysis of stereochemical outcomes is detailed. In the final case, an asymmetric Mannich reaction was developed to install a protected amine directly at the C2 position. Symmetry-breaking reduction of this material gave way to a remarkable series of stereochemical outcomes leading to the title compound without recourse to non-strategic downstream manipulations. This synthesis is immediately accommodating to the facile preparation of structural analogs.

A synthetic strategy to establish five contiguous stereocenters, in a stereocontrolled manner, on the core structure of pactamycin is described. This sequence exploits the use of a Lewis acid mediated epoxide opening cascade to set the relative configuration of the C4–C5 diol while reversing the configuration at C7. This sequence provides the oxygenated core of pactamycin in just 11 steps.

An advanced intermediate in a projected synthesis of pactamycin has been prepared. Early installation of the C1-dimethylurea functionality allows for its participation in a diastereoselective, chelation-controlled addition of organometal nucleophiles to the C5 prochiral ketone. Four of the molecule's six stereocenters are set with a ketone functional handle provided for subsequent manipulation.

A new pactamycin analogue, 5"-fluoropactamycin, was prepared by directed biosynthesis. Supplementation of the fermentation medium of Streptomyces pactum, var. pactum with 3-amino-5-fluorobenzoic acid, an analogue of 3-aminobenzoic acid, an advanced precursor in pactamycin biosynthesis, resulted in co-production of pactamycin and the new pactamycin analogue. A similar feeding experiment with 3-amino-5-methylbenzoic acid did not result in formation of the corresponding methylated pactamycin analogue, but only in inhibition of pactamycin production. Comparison of antimicrobial and cytotoxic activities of pactamycin and 5"-fluoropactamycin showed no significant differences.

The compound is synthesized in 29 linear steps and 3% overall yield starting from a known oxazoline readily available from L-threonine.

Agents, such as EGTA, thapsigargin, and ionophore A23187, that mobilize sequestered Ca2+ from the endoplasmic reticulum (ER) or dithiothreitol (DTT) that compromises the oxidizing environment of the organelle, disrupt early protein processing and inhibit translational initiation. Increased phosphorylation of eIF-2 alpha (5-fold) and inhibition of eIF-2B activity (50%) occur in intact GH3 cells exposed to these agents for 15 min (Prostko et al. J. Biol. Chem. 267:16751-16754, 1992). Alterations in eIF-2 alpha phosphorylation and translational activity in response to EGTA were reversed by addition of Ca2+ in excess of chelator while responses to DTT were reversible by washing. Exposure for 3 h to either A23187 or DTT, previously shown to induce transcription-dependent translational recovery, resulted in dephosphorylation of eIF-2 alpha in a manner blocked by actinomycin D. Phosphorylation of eIF-2 alpha in response to A23187 or DTT was not prevented by conventional inhibitors of translation including cycloheximide, pactamycin, puromycin, or verrucarin. Prolonged inhibition of protein synthesis to deplete the ER of substrates for protein processing resulted in increased eIF-2 alpha phosphorylation, decreased eIF-2B activity, and reduced monosome content that were indicative of time-dependent blockade; these inhibitors did not abolish polysomal content. Superphosphorylation of eIF-2 alpha occurred upon exposure of these preparations to either A23187 or DTT. Tunicamycin, an inhibitor of co-translational transfer of core oligosaccharide, provoked rapid phosphorylation of eIF-2 alpha and inhibition of translational initiation whereas sugar analog inhibitors of glycoprotein processing did neither. A flow of processible protein to the ER does not appear to be required for the phosphorylation of eIF-2 alpha in response to ER perturbants. We hypothesize that perturbation of the translocon, rather than suppression of protein processing, initiates the signal emanating from the ER culminating in eIF-2 alpha phosphorylation and translational repression.

Summary Pactamycin, one of the most densely functionalized aminocyclitol antibiotics, has pronounced antibacterial, antitumor, antiviral, and antiplasmodial activities, but its development as a clinical drug was hampered by its broad cytotoxicity. Efforts to modulate the biological activity by structural modifications using synthetic organic chemistry have been difficult because of the complexity of its chemical structure. However, through extensive biosynthetic studies and genetic engineering, we were able to produce analogs of pactamycin that show potent antimalarial activity, but lack significant antibacterial activity, and are about 10–30 times less toxic than pactamycin toward mammalian cells. The results suggest that distinct ribosomal binding selectivity or new mechanism(s) of action may be involved in their plasmodial growth inhibition, which may lead to the discovery of new antimalarial drugs and identification of new molecular targets within malarial parasites.

A new pactamycin analogue, 5"-fluoropactamycin, was prepared by directed biosynthesis. Supplementation of the fermentation medium of Streptomyces pactum, var. pactum with 3-amino-5-fluorobenzoic acid, an analogue of 3-aminobenzoic acid, an advanced precursor in pactamycin biosynthesis, resulted in co-production of pactamycin and the new pactamycin analogue. A similar feeding experiment with 3-amino-5-methylbenzoic acid did not result in formation of the corresponding methylated pactamycin analogue, but only in inhibition of pactamycin production. Comparison of antimicrobial and cytotoxic activities of pactamycin and 5"-fluoropactamycin showed no significant differences.

This study investigates the capacity of neuronal growth cones to synthesize protein locally and independently of their cell body. Isolated growth cones were prepared from cultures of neurons from the snail Helisoma by transecting neurites proximal to the growth cone. The capacity for protein synthesis was tested by radiolabeling cultures with 3H-leucine and analyzing the resultant autoradiograms. Isolated growth cones displayed incorporation of 3H-leucine that was inhibited by treatment with the protein synthesis inhibitors anisomycin and pactamycin, indicating that ribosomal-dependent translation occurs in growth cones. Ultrastructural analyses of growth cones demonstrated the presence of polyribosomes, the machinery for protein synthesis. The density of polyribosomes varied between growth cones, even between different growth cones on the same neuron, suggesting that growth cones express a range of protein synthetic capabilities. That different types of growth cones possess differing capabilities for protein synthesis is suggested in autoradiograms of 3H-leucine incorporation by the growth cones of axonal and nonaxonal neurites; incorporation was radically reduced in axonal growth cones in comparison with non-axonal growth cones. Finally, growth cones that were isolated for 2 d prior to radiolabeling incorporate 3H-leucine in a eukaryotic ribosomal- dependent manner, suggesting that the capacity for translation is long- lived in growth cones. Taken together, these studies reveal a capacity for protein synthesis confined totally to the neuronal growth cone proper. The synthesis of proteins in growth cones could afford a mechanism for the alteration of growth cone structure or function. This is in accord with the view that growth cones participate autonomously, to at least some extent, in the processes of synaptogenesis and the construction of neuronal architecture.

The role of protein synthesis in the control of phosphoenolpyruvate carboxykinase (PEPCK; 4.1.1.32) mRNA turnover was studied in FTO-2B rat hepatoma cells. A previous study demonstrated that incubation of these cells with cAMP prolongs the half-life of the otherwise short-lived PEPCK mRNA. The decay rate of PEPCK mRNA was also slowed in cells incubated with cycloheximide, but not in cells incubated with other translation inhibitors, such as puromycin or pactamycin, even though protein synthesis was inhibited 85-95% by these agents. No correlation was noted between the rate of L-[3H]valine incorporation into cellular proteins and PEPCK mRNA half-life, suggesting that protein synthesis per se is not required for breakdown of the mRNA. Exposure of cells to the translation initiation inhibitor pactamycin together with cycloheximide abolished the "slowing" effect of cycloheximide, and PEPCK mRNA decayed at the same rate as in cells incubated in the presence of pactamycin alone. In contrast, pactamycin did not reverse the effect of cAMP, and the mRNA decayed at the same slow rate in cells incubated in the presence of either (Bu)2cAMP alone or (Bu)2cAMP together with pactamycin. Since pactamycin promotes polysomes dissociation, these results suggest that cAMP enhances the stability of a polysome-free PEPCK mRNA. Furthermore, these results strongly indicate that neither the rapid decay of PEPCK mRNA nor the cAMP-mediated stabilization of the mRNA requires on-going protein synthesis.

Ribosomal resistance to pactamycin in clones of Streptomyces lividans containing DNA (pct) from Streptomyces pactum, the pactamycin producer, involves methylation of 16S RNA. The modified residue A-941 in S. lividans 16S rRNA (A-964 in the homologous Escherichia coli sequence) is converted to 1-methyladenosine, and the ribosomal ability to bind pactamycin is reduced or abolished.

We have studied the interactions of the antibiotics apramycin, kasugamycin, myomycin, neamine and pactamycin with 16S rRNA by chemical probing of drug-ribosome complexes. Kasugamycin and pactamycin, which are believed to affect translational initiation, protect bases in common with P-site-bound tRNA. While kasugamycin protects A794 and G926, and causes enhanced reactivity of C795, pactamycin protects G693 and C795. All four of these bases were previously shown to be protected by P-site tRNA or by edeine, another P-site inhibitor. Apramycin and neamine, which both induce miscoding and inhibit translocation, protect A1408, G1419 and G1494, as was also found earlier for neomycin, gentamicin, kanamycin and paromomycin. A1408 and G1494 were previously shown to be protected by A-site tRNA. Surprisingly, myomycin fails to give strong protection of any bases in 16S rRNA, in spite of having an apparently identical target site and mode of action to streptomycin, which protects several bases in the 915 region. Instead, myomycin gives only weak protection of A1408. These results suggest that the binding site(s) of streptomycin and myomycin have yet to be identified.

Natural products—small molecules isolated from plants, fungi, bacteria, and other microorganisms—continue to serve as an important source of chemical tools for the study of biological systems and disease pathology, as well as new drugs. One example is the natural product pactamycin, which has been instrumental in the investigation of ribosome structure and function (1, 2). However, the structural complexity of this small molecule has historically rendered it—and by extension its unnatural analogs—synthetically inaccessible, hindering efforts at the development of pactamycin-derived therapeutics. On page 180 of this issue, Malinowski et al. (3) report a total chemical synthesis of pactamycin that elegantly addresses this challenge and opens a new chapter in the story of this natural product

The lambda cI lysogenic transcript is unusual in having no leader. Expression of a cI-lacZ protein fusion was relatively resistant to kasugamycin and pactamycin, which inhibit translation initiation on transcripts with leaders. Our data imply that there are distinct differences in translation initiation between the two classes of transcripts.

The iron-induced biosynthesis of ferritin is regulated at the translational level via multiple mechanism. A prolonged exposure of cells to iron leads to a marked increase in ferritin mRNA levels caused by stabilization of the message. Here we show that this stabilization requires the synthesis de novo of an iron-inducible protein factor.