Translational PK/PD for the Development of Novel Antibiotics-A Drug Developer's Perspective.

Antibiotic development traditionally involved large Phase 3 programs, preceded by Phase 2 studies. Recognizing the high unmet medical need for new antibiotics and, in some cases, challenges to conducting large clinical trials, regulators created a streamlined clinical development pathway in which a lean clinical efficacy dataset is complemented by nonclinical data as supportive evidence of efficacy. In this context, translational Pharmacokinetic/Pharmacodynamic (PK/PD) plays a key role and is a major contributor to a "robust" nonclinical package. The classical PK/PD index approach, proven successful for established classes of antibiotics, is at the core of recent antibiotic approvals and the current antibacterial PK/PD guidelines by regulators. Nevertheless, in the case of novel antibiotics with a novel Mechanism of Action (MoA), there is no prior experience with the PK/PD index approach as the basis for translating nonclinical efficacy to clinical outcome, and additional nonclinical studies and PK/PD analyses might be considered to increase confidence. In this review, we discuss the value and limitations of the classical PK/PD approach and present potential risk mitigation activities, including the introduction of a semi-mechanism-based PK/PD modeling approach. We propose a general nonclinical PK/PD package from which drug developers might choose the studies most relevant for each individual candidate in order to build up a "robust" nonclinical PK/PD understanding.

Improving the ability of antimicrobial susceptibility tests to predict clinical outcome accurately: Adding metabolic evasion to the equation.

Antimicrobial susceptibility tests (AST) are based on the minimal inhibitory concentration (MIC), the method used worldwide to guide antimicrobial therapy. Despite its relevance in correctly predicting clinical outcome for most acute infections, this approach is misleading for multiple clinical cases in which pathogens do not grow rapidly, uniformly or with physical protection. This behaviour, named 'metabolic evasion' (ME), enables bacteria to survive antimicrobials. ME can result from different, and sometimes combined, bacterial mechanisms such as biofilms, intracellular growth, persisters or dormancy. We discuss how ME can influence the MIC-based probability of target attainment. We identify clinical cases in which this approach is undermined by ME and propose a new approach that takes ME into account in order to improve patient management and the evaluation of innovative drugs.

Afabicin, a First-in-Class Antistaphylococcal Antibiotic, in the Treatment of Acute Bacterial Skin and Skin Structure Infections: Clinical Noninferiority to Vancomycin/Linezolid.

Afabicin (formerly Debio 1450, AFN-1720) is a prodrug of afabicin desphosphono, an enoyl-acyl carrier protein reductase (FabI) inhibitor, and is a first-in-class antibiotic with a novel mode of action to specifically target fatty acid synthesis in Staphylococcus spp. The efficacy, safety, and tolerability of afabicin were compared with those of vancomycin/linezolid in the treatment of acute bacterial skin and skin structure infections (ABSSSI) due to staphylococci in this multicenter, parallel-group, double-blind, and double-dummy phase 2 study. Randomized patients (1:1:1) received either low-dose (LD) afabicin (intravenous [i.v.] 80 mg, followed by oral 120 mg, twice a day [BID]), high-dose (HD) afabicin (i.v. 160 mg, followed by oral 240 mg, BID), or vancomycin/linezolid (i.v. vancomycin 1 g or 15 mg/kg, followed by oral linezolid 600 mg, BID). The most frequent baseline pathogen was Staphylococcus aureus (97.5% of microbiological intent-to-treat [mITT] population), and 50.4% of patients had methicillin-resistant S. aureus Clinical response rates at 48 to 72 h postrandomization in the mITT population were comparable among treatment groups (94.6%, 90.1%, and 91.1%, respectively). Both LD and HD afabicin were noninferior to vancomycin/linezolid (differences, -3.5% [95% confidence interval {CI}, -10.8%, 3.9%] and 1.0% [95% CI, -7.3%, 9.2%], respectively). Most common treatment-emergent adverse events were mild and were headache (9.1% and 16.8%) and nausea (6.4% and 8.4%) with LD and HD afabicin, respectively. Afabicin was efficacious and well tolerated in the treatment of ABSSSI due to staphylococci, and these data support further development of afabicin for the treatment of ABSSSI and potentially other types of staphylococcal infections. (This study has been registered at ClinicalTrials.gov under identifier NCT02426918.).

Cellular pharmacokinetics and intracellular activity of the bacterial fatty acid synthesis inhibitor, afabicin desphosphono against different resistance phenotypes of Staphylococcus aureus in models of cultured phagocytic cells.

Antibiotics with new modes of action that are active against intracellular forms of Staphylococcus aureus are sorely needed to fight recalcitrant infections caused by this bacterium. Afabicin desphosphono (Debio 1452, the active form of afabicin [Debio 1450]) is an inhibitor of FabI enoyl-Acyl carrier protein reductase and has specific and extremely potent activity against Staphylococci, including strains resistant to current antistaphylococcal agents. Using mouse J774 macrophages and human THP-1 monocytes, we showed that afabicin desphosphono: (i) accumulates rapidly in cells, reaching stable cellular-to-extracellular concentration ratios of about 30; (ii) is recovered entirely and free in the cell-soluble fraction (no evidence of stable association with proteins or other macromolecules). Afabicin desphosphono caused a maximum cfu decrease of about 2.5 log10 after incubation in broth for 30 h, including against strains resistant to vancomycin, daptomycin, and/or linezolid. Using a pharmacodynamic model of infected THP-1 monocytes (30 h of incubation post-phagocytosis), we showed that afabicin desphosphono is bacteriostatic (maximum cfu decrease: 0.56 to 0.73 log10) towards all strains tested, a behaviour shared with the comparators (vancomycin, daptomycin, and linezolid) when tested against susceptible strains. We conclude that afabicin desphosphono has a similar potential as vancomycin, daptomycin or linezolid to control the intracellular growth and survival of phagocytized S. aureus and remains fully active against strains resistant to these comparators.

Sputum containing zinc enhances carbapenem resistance, biofilm formation and virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa chronic lung infections are the leading cause of mortality in cystic fibrosis patients, a serious problem which is notably due to the numerous P. aeruginosa virulence factors, to its ability to form biofilms and to resist the effects of most antibiotics. Production of virulence factors and biofilm formation by P. aeruginosa is highly coordinated through complex regulatory systems. We recently found that CzcRS, the zinc and cadmium-specific two-component system is not only involved in metal resistance, but also in virulence and carbapenem antibiotic resistance in P. aeruginosa. Interestingly, zinc has been shown to be enriched in the lung secretions of cystic fibrosis patients. In this study, we investigated whether zinc might favor P. aeruginosa pathogenicity using an artificial sputum medium to mimic the cystic fibrosis lung environment. Our results show that zinc supplementation triggers a dual P. aeruginosa response: (i) it exacerbates pathogenicity by a CzcRS two-component system-dependent mechanism and (ii) it stimulates biofilm formation by a CzcRS-independent mechanism. Furthermore, P. aeruginosa cells embedded in these biofilms exhibited increased resistance to carbapenems. We identified a novel Zn-sensitive regulatory circuit controlling the expression of the OprD porin and modifying the carbapenem resistance profile. Altogether our data demonstrated that zinc levels in the sputum of cystic fibrosis patients might aggravate P. aeruginosa infection. Targeting zinc levels in sputum would be a valuable strategy to curb the increasing burden of P. aeruginosa infections in cystic fibrosis patients.

The nuclear pore regulates GAL1 gene transcription by controlling the localization of the SUMO protease Ulp1.

Transcription activation of some yeast genes correlates with their repositioning to the nuclear pore complex (NPC). The NPC-bound Mlp1 and Mlp2 proteins have been shown to associate with the GAL1 gene promoter and to maintain Ulp1, a key SUMO protease, at the NPC. Here, we show that the release of Ulp1 from the NPC increases the kinetics of GAL1 derepression, whereas artificial NPC anchoring of Ulp1 in the Δmlp1/2 strain restores normal GAL1 regulation. Moreover, artificial tethering of the Ulp1 catalytic domain to the GAL1 locus enhances the derepression kinetics. Our results also indicate that Ulp1 modulates the sumoylation state of Tup1 and Ssn6, two regulators of glucose-repressed genes, and that a loss of Ssn6 sumoylation correlates with an increase in GAL1 derepression kinetics. Altogether, our data highlight a role for the NPC-associated SUMO protease Ulp1 in regulating the sumoylation of gene-bound transcription regulators, positively affecting transcription kinetics in the context of the NPC.

The transcriptional regulator CzcR modulates antibiotic resistance and quorum sensing in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa responds to zinc, cadmium and cobalt by way of the CzcRS two-component system. In presence of these metals the regulatory protein CzcR induces the expression of the CzcCBA efflux pump, expelling and thereby inducing resistance to Zn, Cd and Co. Importantly, CzcR co-regulates carbapenem antibiotic resistance by repressing the expression of the OprD porin, the route of entry for these antibiotics. This unexpected co-regulation led us to address the role of CzcR in other cellular processes unrelated to the metal response. We found that CzcR affected the expression of numerous genes directly involved in the virulence of P. aeruginosa even in the absence of the inducible metals. Notably the full expression of quorum sensing 3-oxo-C12-HSL and C4-HSL autoinducer molecules is impaired in the absence of CzcR. In agreement with this, the virulence of the czcRS deletion mutant is affected in a C. elegans animal killing assay. Additionally, chromosome immunoprecipitation experiments allowed us to localize CzcR on the promoter of several regulated genes, suggesting a direct control of target genes such as oprD, phzA1 and lasI. All together our data identify CzcR as a novel regulator involved in the control of several key genes for P. aeruginosa virulence processes.

Connecting the transcription site to the nuclear pore: a multi-tether process that regulates gene expression.

It is now well established that the position of a gene within the nucleus can influence the level of its activity. So far, special emphasis has been placed on the nuclear envelope (NE) as a transcriptionally silent nuclear sub-domain. Recent work, however, indicates that peripheral localization is not always associated with repression, but rather fulfills a dual function in gene expression. In particular, in the yeast Saccharomyces cerevisiae, a large number of highly expressed genes and activated inducible genes preferentially associate with nuclear pore complexes (NPCs), a process that is mediated by transient interactions between the transcribed locus and the NPC. Recent studies aimed at unraveling the molecular basis of this mechanism have revealed that maintenance of genes at the NPC involves multiple tethers at different steps of gene expression. These observations are consistent with tight interconnections between transcription, mRNA processing and export into the cytoplasm, and highlight a role for the NPC in promoting and orchestrating the gene expression process. In this Commentary, we discuss the factors involved in active gene anchoring to the NPC and the diverse emerging roles of the NPC environment in promoting gene expression, focusing on yeast as a model organism.

THO/Sub2p functions to coordinate 3'-end processing with gene-nuclear pore association.

During transcription, proteins assemble sequentially with nascent RNA to generate a messenger ribonucleoprotein particle (mRNP). The THO complex and its associated Sub2p helicase are functionally implicated in both transcription and mRNP biogenesis but their precise function remains elusive. We show here that THO/Sub2p mutation leads to the accumulation of a stalled intermediate in mRNP biogenesis that contains nuclear pore components and polyadenylation factors in association with chromatin. Microarray analyses of genomic loci that are aberrantly docked to the nuclear pore in mutants allowed the identification of approximately 400 novel validated target genes that require THO /Sub2p for efficient expression. Our data strongly suggests that the THO complex/Sub2p function is required to coordinate events leading to the acquisition of export competence at a step that follows commitment to 3'-processing.

Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae.

Genome-wide studies in S. cerevisiae reveal that the transcriptome includes numerous antisense RNAs as well as intergenic transcripts regulated by the exosome component Rrp6. We observed that upon the loss of Rrp6 function, two PHO84 antisense transcripts are stabilized, and PHO84 gene transcription is repressed. Interestingly, the same phenotype is observed in wild-type cells during chronological aging. Epistasis and chromatin immunoprecipitation experiments indicate that the loss of Rrp6 function is paralleled by the recruitment of Hda1 histone deacetylase to PHO84 and neighboring genes. However, histone deacetylation is restricted to PHO84, suggesting that Hda1 activity depends on antisense RNA. Accordingly, the knockdown of antisense production prevents PHO84 gene repression, even in the absence of Rrp6. Together, our data indicate that the stabilization of antisense transcripts results in PHO84 gene repression via a mechanism distinct from transcription interference and that the modulation of Rrp6 function contributes to gene regulation by inducing RNA-dependent epigenetic modifications.

Cotranscriptional recruitment to the mRNA export receptor Mex67p contributes to nuclear pore anchoring of activated genes.

Transcription activation of some Saccharomyces cerevisiae genes is paralleled by their repositioning to the nuclear periphery, but the mechanism underlying gene anchoring is poorly defined. We show that the nuclear pore complex-associated Mlp1p and the shuttling mRNA export receptor Mex67p contribute to the stable association of the activated GAL10 and HSP104 genes with the nuclear periphery. However, we find no obligatory link between gene positioning and gene expression. Furthermore, gene anchoring correlates with the cotranscriptional recruitment of Mex67p to transcribing genes. Notably, the association of Mex67p with chromatin is not mediated by RNA. Interestingly, a mutant GAL2 gene lacking the coding region is still able to recruit Mex67p upon transcriptional activation and to relocate to the nuclear periphery. Together these data suggest that, at least for GAL2, nascent messenger ribonucleoprotein does not play a major role in gene anchoring and that the early recruitment of Mex67p contributes to gene repositioning by virtue of an RNA-independent process.