Peptide Targeting of an Antibiotic Prodrug toward Phagosome-Entrapped Mycobacteria.

Mycobacterial infections are difficult to treat due to the bacterium's slow growth, ability to reside in intracellular compartments within macrophages, and resistance mechanisms that limit the effectiveness of conventional antibiotics. Developing antibiotics that overcome these challenges is therefore critical to providing a pipeline of effective antimicrobial agents. Here, we describe the synthesis and testing of a unique peptide-drug conjugate that exhibits high levels of antimicrobial activity against M. smegmatis and M. tuberculosis as well as clearance of intracellular mycobacteria from cultured macrophages. Using an engineered peptide sequence, we deliver a potent DHFR inhibitor and target the intracellular phagosomes where mycobacteria reside and also incorporate a ß-lactamase-cleavable cephalosporin linker to enhance the targeting of quiescent intracellular ß-lactam-resistant mycobacteria. By using this type of prodrug approach to target intracellular mycobacterial infections, the emergence of antibacterial resistance mechanisms could be minimized.

Structural modifications of mitochondria-targeted chlorambucil alter cell death mechanism but preserve MDR evasion.

Multidrug resistance (MDR) remains one of the major obstacles in chemotherapy, potentially rendering a multitude of drugs ineffective. Previously, we have demonstrated that mitochondrial targeting of DNA damaging agents is a promising tool for evading a number of common resistance factors that are present in the nucleus or cytosol. In particular, mitochondria-targeted chlorambucil (mt-Cbl) has increased potency and activity against resistant cancer cells compared to the parent compound chlorambucil (Cbl). However, it was found that, due to its high reactivity, mt-Cbl induces a necrotic type of cell death via rapid nonspecific alkylation of mitochondrial proteins. Here, we demonstrate that by tuning the alkylating activity of mt-Cbl via chemical modification, the rate of generation of protein adducts can be reduced, resulting in a shift of the cell death mechanism from necrosis to a more controlled apoptotic pathway. Moreover, we demonstrate that all of the modified mt-Cbl compounds effectively evade MDR resulting from cytosolic GST-µ upregulation by rapidly accumulating in mitochondria, inducing cell death directly from within. In this study, we systematically elucidated the advantages and limitations of targeting alkylating agents with varying reactivity to mitochondria.

Molecular vehicles for mitochondrial chemical biology and drug delivery.

The mitochondria within human cells play a major role in a variety of critical processes involved in cell survival and death. An understanding of mitochondrial involvement in various human diseases has generated an appreciable amount of interest in exploring this organelle as a potential drug target. As a result, a number of strategies to probe and combat mitochondria-associated diseases have emerged. Access to mitochondria-specific delivery vectors has allowed the study of biological processes within this intracellular compartment with a heightened level of specificity. In this review, we summarize the features of existing delivery vectors developed for targeting probes and therapeutics to this highly impermeable organelle. We also discuss the major applications of mitochondrial targeting of bioactive molecules, which include the detection and treatment of oxidative damage, combating bacterial infections, and the development of new therapeutic approaches for cancer. Future directions include the assessment of the therapeutic benefit achieved by mitochondrial targeting for treatment of disease in vivo. In addition, the availability of mitochondria-specific chemical probes will allow the elucidation of the details of biological processes that occur within this cellular compartment.

Targeting mitochondrial DNA with a platinum-based anticancer agent.

An analog of the anticancer drug cisplatin (mtPt) was delivered to mitochondria of human cells using a peptide specifically targeting this organelle. mtPt induces apoptosis without damaging nuclear DNA, indicating that mtDNA damage is sufficient to mediate the activity of a platinum-based chemotherapeutic. This study demonstrates the specific delivery of a platinum drug to mitochondria and investigates the effects of directing this agent outside the nucleus.

Re-directing an alkylating agent to mitochondria alters drug target and cell death mechanism.

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.

Tuning the activity of mitochondria-penetrating peptides for delivery or disruption.

Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.

Rerouting chlorambucil to mitochondria combats drug deactivation and resistance in cancer cells.

The difficulty of accessing the mitochondrial matrix has limited the targeting of therapeutics to this organelle. Here, we report, to our knowledge, the first successful delivery of an active DNA alkylating agent--chlorambucil--to mitochondria, and describe unexpected features that result from rerouting this drug within the cell. Mitochondrial targeting of this agent dramatically potentiates its activity, and promotes apoptotic cell death in a variety of cancer cell lines and patient samples. This retention of activity is observed even in cells with resistance to chlorambucil or disabled apoptotic triggering.

Maximizing the therapeutic window of an antimicrobial drug by imparting mitochondrial sequestration in human cells.

The number of antimicrobial agents available for use in humans is limited by the difficulty of discovering chemical agents with selective toxicity to bacterial targets. Numerous small molecule inhibitors have potential as antimicrobial agents, yet their use has been prevented by high levels of toxic cross-reactivity in human cells. For example, methotrexate (Mtx) is an effective antimetabolite that exerts its effects by inhibiting DHFR. It is a potent antibacterial when accumulated intracellularly, but toxicity in human cells limits clinical utility in infectious disease treatment. Here, we describe peptide conjugates of Mtx that are sequestered into the mitochondria of human cells (mt-Mtx). This alteration in localization of Mtx, which directs it away from its enzyme target, decreases its toxicity in human cells by a factor of 10(3). Mt-Mtx, however, maintains activity against a variety of pathogenic gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA). The results from this proof-of-principle study describe a novel methodology for augmenting the antibacterial efficacy of drugs amenable to peptide conjugation while simultaneously decreasing their toxicity to the host organism.

Are essential genes really essential?

Gene essentiality has emerged as an often-asked question in the wake of bacterial genome sequencing and a renaissance in studies of prokaryotic physiology. Genome-scale efforts at describing essential gene sets have necessarily been carried out under standard and tractable growth conditions in a laboratory setting. In addition to reinforcing our understanding of core bacterial physiology, these studies have also uncovered large numbers of essential genes encoding proteins whose functions remain poorly described. Studies of these and other elements of core physiology have naturally followed and several paradoxes, relating to growth conditions and genetic context, have begun to challenge our understanding of the term "essential gene". Most recently genome-scale genetic interaction studies have revealed remarkable density and redundancy in biological systems with profound implications for dispensability phenotypes associated with single gene mutations. Consequently, the phenotype "essential" should be carefully viewed as contextual.

The wall teichoic acid polymerase TagF is non-processive in vitro and amenable to study using steady state kinetic analysis.

Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid , to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid analog species varying in size have the same K(m) value of 2.6 microm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.

Recent advances in the use of cell-penetrating peptides for medical and biological applications.

The selective permeability of the plasma membrane prohibits most exogenous agents from gaining cellular access. Since many therapeutics and reporter molecules must be internalized for activity, crossing the plasma membrane is essential. A very effective class of transporters harnessed for this purpose are cell penetrating peptides (CPPs), a group of short cationic sequences with a remarkable capacity for membrane translocation. Since their discovery in 1988, CPPs have been employed for the delivery of a wide variety of cargo including small molecules, nucleic acids, antibodies and nanoparticles. This review describes recent advances in the use of CPPs for biological and therapeutic applications. In particular, an emphasis is placed on novel systems and insights acquired since 2006. Basic research on CPPs has recently yielded techniques that provide further information on the controversial mechanism of CPP uptake and has also resulted in the development of new model membrane systems to evaluate these mechanisms. In addition, recent use of CPPs for the development of new cellular imaging tools, biosensors, or biomolecular delivery systems have been highlighted. Lastly, novel peptide delivery vectors, designed to tackle some of the drawbacks of CPPs and enhance their versatility, will be described. This review will illustrate the diverse applications for which CPPs have been harnessed and also demonstrate the remarkable advancements these peptides have facilitated in cell biology.

High-throughput screening identifies novel inhibitors of the acetyltransferase activity of Escherichia coli GlmU.

The bifunctional GlmU protein catalyzes the formation of UDP-N-acetylglucosamine in a two-step reaction using the substrates glucosamine-1-phosphate, acetyl coenzyme A, and UTP. This metabolite is a common precursor to the synthesis of bacterial cell surface carbohydrate polymers, such as peptidoglycan, lipopolysaccharide, and wall teichoic acid that are involved in the maintenance of cell shape, permeability, and virulence. The C-terminal acetyltransferase domain of GlmU exhibits structural and mechanistic features unique to bacterial UDP-N-acetylglucosamine synthases, making it an excellent target for antibacterial design. In the work described here, we have developed an absorbance-based assay to screen diverse chemical libraries in high throughput for inhibitors to the acetyltransferase reaction of Escherichia coli GlmU. The primary screen of 50,000 drug-like small molecules identified 63 hits, 37 of which were specific to acetyltransferase activity of GlmU. Secondary screening and mode-of-inhibition studies identified potent inhibitors where compound binding within the acetyltransferase active site was requisite on the presence of glucosamine-1-phosphate and were competitive with the substrate acetyl coenzyme A. These molecules may represent novel chemical scaffolds for future antimicrobial drug discovery. In addition, this work outlines the utility of catalytic variants in targeting specific activities of bifunctional enzymes in high-throughput screens.

Duplication of teichoic acid biosynthetic genes in Staphylococcus aureus leads to functionally redundant poly(ribitol phosphate) polymerases.

Wall teichoic acids are anionic phosphate-rich polymers that are part of the complex meshwork of carbohydrates that make up the gram-positive cell wall. These polymers are essential to the proper rod-shaped morphology of Bacillus subtilis and have been shown to be an important virulence determinant in the nosocomial opportunistic pathogen Staphylococcus aureus. Together, sequence-based studies, in vitro experiments with biosynthetic proteins, and analyses of the chemical structure of wall teichoic acid have begun to shed considerable light on our understanding of the biogenesis of this polymer. Nevertheless, some paradoxes remain unresolved. One of these involves a putative duplication of genes linked to CDP-ribitol synthesis (tarI'J' and tarIJ) as well as poly(ribitol phosphate) polymerization (tarK and tarL) in S. aureus. In the work reported here, we performed careful studies of the dispensability of each gene and discovered a functional redundancy in the duplicated gene clusters. We were able to create mutants in either of the putative ribitol phosphate polymerases (encoded by tarK and tarL) without affecting teichoic acid levels in the S. aureus cell wall. Although genes linked to CDP-ribitol synthesis are also duplicated, a null mutant in only one of these (tarI'J') could be obtained, while tarIJ remained essential. Suppression analysis of the tarIJ null mutant indicated that the mechanism of dysfunction in tarI'J' is due to poor translation of the TarJ' enzyme, which catalyzes the rate-limiting step in CDP-ribitol formation. This work provides new insights into understanding the complex synthetic steps of the ribitol phosphate polymer in S. aureus and has implications on specifically targeting enzymes involved in polymer biosynthesis for antimicrobial design.

Lesions in teichoic acid biosynthesis in Staphylococcus aureus lead to a lethal gain of function in the otherwise dispensable pathway.

An extensive study of teichoic acid biosynthesis in the model organism Bacillus subtilis has established teichoic acid polymers as essential components of the gram-positive cell wall. However, similar studies pertaining to therapeutically relevant organisms, such as Staphylococcus aureus, are scarce. In this study we have carried out a meticulous examination of the dispensability of teichoic acid biosynthetic enzymes in S. aureus. By use of an allelic replacement methodology, we examined all facets of teichoic acid assembly, including intracellular polymer production and export. Using this approach we confirmed that the first-acting enzyme (TarO) was dispensable for growth, in contrast to dispensability studies in B. subtilis. Upon further characterization, we demonstrated that later-acting gene products (TarB, TarD, TarF, TarIJ, and TarH) responsible for polymer formation and export were essential for viability. We resolved this paradox by demonstrating that all of the apparently indispensable genes became dispensable in a tarO null genetic background. This work suggests a lethal gain-of-function mechanism where lesions beyond the initial step in wall teichoic acid biosynthesis render S. aureus nonviable. This discovery poses questions regarding the conventional understanding of essential gene sets, garnered through single-gene knockout experiments in bacteria and higher organisms, and points to a novel drug development strategy targeting late steps in teichoic acid synthesis for the infectious pathogen S. aureus.

Bifunctional catalysis by CDP-ribitol synthase: convergent recruitment of reductase and cytidylyltransferase activities in Haemophilus influenzae and Staphylococcus aureus.

CDP-ribitol synthase catalyzes the formation of CDP-ribitol from ribulose 5-phosphate, NADPH, and CTP. CDP-ribitol is an activated precursor for the synthesis of virulence-associated polysaccharides in the capsule of the Gram-negative pathogen Haemophilus influenzae and in the cell walls of Gram-positive pathogens including Staphylococcus aureus. We showed previously that CDP-ribitol synthase activity in H. influenzae is catalyzed by the bifunctional enzyme Bcs1 in a two-step reaction with reduction preceding cytidylyl transfer [Zolli, M., et al. (2001) Biochemistry 40, 5041-5048]. In the work reported here, we predicted a CDP-ribitol synthesis locus in S. aureus tandemly arranged as tarI, encoding an orthologue of the cytidylyltransferase domain of Bcs1, and tarJ, coding for an analogue of the reductase domain of Bcs1. We have shown the formation of a functional CDP-ribitol synthase complex between TarI and TarJ. Steady-state mechanistic studies of the CDP-ribitol synthases TarIJ and Bcs1 revealed that the analogous reductases and orthologous cytidylyltransferases undergo ordered mechanisms. The sequence of substrate binding and product release of the orthologous cytidylyltransferases differed. Steady-state analysis of the reductase and cytidylyltransferase activities of TarIJ indicated a 100-fold difference in the turnover where the primary reductase was rate limiting. Rapid mixing experiments revealed the presence of approximately 12 microM ribitol 5-phosphate at steady state, 100-fold lower than the observed K(m) for this intermediate. Analysis of the approach to steady state suggested that channeling was not occurring in the coupled enzyme complex and was an unlikely driving force in the convergent recruitment of reductase and cytidylyltransferase activities in the two CDP-ribitol synthases.