Hitting "Undruggable" Targets: Determining the Properties of Cell Penetrant Stabilized Peptide Therapeutics for Intracellular Targets

Nearly two-thirds of all disease-associated proteins are ‘undruggable’ by modern therapeutics, meaning they are inside cells, out of the reach of biologics, but lack small molecule binding pockets. Stabilized peptides have the potential to hit these targets, which would open a vast array of potential new therapies. One such target is the p53/MDM2 interaction—a protein-protein interaction central to many cancers. Several inhibitors have been developed against the MDM2 protein because this target degrades the “the guardian of the genome” protein, p53. However, few of these peptides demonstrate the serum-independent, on-target efficacy required for clinical translation. In addition to having a strong target binding affinity, these peptides must efficiently penetrate the cell and evade proteolytic degradation. The design criteria for developing agents that can meet all of these requirements are still poorly understood. This work focuses on identifying the most important physicochemical properties that promote overall in vitro efficacy, taking into account the relevant molecular and cellular parameters in order to aid in future design of stabilized peptide therapeutics. The research presented here begins with measuring the effects that lipophilicity and charge have on have on cellular uptake as these are two commonly tuned parameters for promoting stabilized peptide efficacy. Furthermore, cellular membrane penetration is largely thought to be a major limiting factor for this class of drugs. Results showed that incremental increases in charge caused significant increases in uptake and that although lipophilic peptides are more efficient at entering cells than hydrophilic peptides, there is a point at which increased lipophilicity begins to instead decrease uptake (logD>~3.5). After obtaining these results, I moved on to selecting peptides discovered via bacterial surface display and measuring their binding affinities and in vitro efficacies as a precursor to a full physicochemical property profile in order to identify what the biggest contributors to efficacy are. After selecting those peptides, I measured their lipophilicities, cellular penetration rates, membrane interactions, and proteolytic stability. Ultimately, results showed that the cellular potency of this series of compounds appears to be driven by intracellular stability, which correlated with efficacy, rather than permeability, which did not at all correlate with efficacy. This was demonstrated by ATSP-7041, a promising MDM2/p53 inhibiting peptide and the only p53-based peptide that has led to a clinical lead compound, as well as pepC, a novel peptide with efficacy close to that of ATSP-7041. These two peptides showed the highest resistance to proteolytic degradation as well as the highest cellular potency, although ATSP-7041 had the slowest cellular uptake (~3-fold slower than pepC). Characterization of the molecules demonstrated they all had high affinity and modest membrane permeability, leading to stability as the differentiating factor. These results exhibit the need for a wholistic assessment of peptide properties to help inform efficacy outcomes and serve as a basis for future peptide development.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/163128/1/atangcho_1.pd

Monoclonal Antibody Production Via Fluidized Bioreactor Technology

Monoclonal antibodies (mAbs) are biologically identical antibodies created by homogenous immune cells originating from the same parent cell. MAbs target a specific epitope of an antigen on a cell’s surface, allowing it to neutralize the antigen. This unique characteristic has made them a key tool in the biopharmaceutical industry for the production of therapeutic drugs. One of these drugs is Rituxan® (rituximab), a mAb drug for the treatment of various cancers and autoimmune diseases. Currently, most mAb products are grown via cell suspension technology in stirred tank bioreactors. However, we have found that by using an integrated bioprocessing model, including conventional cell suspension culture tanks and fluidized bioreactor technology, overall product yield per day is increased by about 7-fold for the production of Rituxan®. Additionally, an economic analysis shows the fluidized bioreactor process is more profitable. Furthermore, though it requires a higher initial investment than the stirred tank process, the differential present worth of the fluidized bioreactor process in comparison to the stirred tank process is $13 billion. Overall, for the production of Rituxan®, the use of fluidized bioreactor technology is a more productive and lucrative process than the conventional stirred tank process

Selective proteomic analysis of antibiotic-tolerant cellular subpopulations in pseudomonas aeruginosa biofilms

Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance

Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms

Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts

Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms

Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts

Structure-Guided Design and Initial Studies of a Bifunctional MEK/PI3K Inhibitor (ST-168)

The structure-based design of a new single entity, MEK/PI3K bifunctional inhibitor (<b>7</b>, <b>ST-168</b>), which displays improved MEK1 and PI3K isoform inhibition, is described. <b>ST-168</b> demonstrated a 2.2-fold improvement in MEK1 inhibition and a 2.8-, 2.7-, 23-, and 2.5-fold improved inhibition toward the PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ isoforms, respectively, as compared to a previous lead compound (<b>4</b>; <b>ST-162</b>) in <i>in vitro</i> enzymatic inhibition assays. <b>ST-168</b> demonstrated superior tumoricidal efficacy over <b>ST-162</b> in an A375 melanoma spheroid tumor model. <b>ST-168</b> was comparatively more effective than <b>ST-162</b> in promoting tumor control when administrated orally in a tumor therapy study conducted in an A375 melanoma mouse model confirming its bioavailability and efficacy toward combined <i>in vivo</i> MEK1/PI3K inhibition