Survival in macrophages induces enhanced virulence in Cryptococcus.

Cryptococcus is a ubiquitous environmental fungus and frequent colonizer of human lungs. Colonization can lead to diverse outcomes, from clearance to long-term colonization to life-threatening meningoencephalitis. Regardless of the outcome, the process starts with an encounter with phagocytes. Using the zebrafish model of this infection, we have noted that cryptococcal cells first spend time inside macrophages before they become capable of pathogenic replication and dissemination. What "licensing" process takes place during this initial encounter, and how are licensed cryptococcal cells different? To address this, we isolated cryptococcal cells after phagocytosis by cultured macrophages and found these macrophage-experienced cells to be markedly more virulent in both zebrafish and mouse models. Despite producing a thick polysaccharide capsule, they were still subject to phagocytosis by macrophages in the zebrafish. Analysis of antigenic cell wall components in these licensed cells demonstrated that components of mannose and chitin are more available for staining than they are in culture-grown cells or cells with capsule production induced in vitro. Cryptococcus is capable of exiting or transferring between macrophages in vitro, raising the likelihood that this fungus alternates between intracellular and extracellular life during growth in the lungs. Our results raise the possibility that intracellular life has its advantages over time, and phagocytosis-induced alteration in mannose and chitin exposure is one way that makes subsequent rounds of phagocytosis more beneficial to the fungus.IMPORTANCECryptococcosis begins in the lungs and can ultimately travel through the bloodstream to cause devastating infection in the central nervous system. In the zebrafish model, small amounts of cryptococcus inoculated into the bloodstream are initially phagocytosed and become far more capable of dissemination after they exit macrophages. Similarly, survival in the mouse lung produces cryptococcal cell types with enhanced dissemination. In this study, we have evaluated how phagocytosis changes the properties of Cryptococcus during pathogenesis. Macrophage-experienced cells (MECs) become "licensed" for enhanced virulence. They out-disseminate culture-grown cells in the fish and out-compete non-MECs in the mouse lung. Analysis of their cell surface demonstrates that MECs have increased availability of cell wall components mannose and chitin substances involved in provoking phagocytosis. These findings suggest how Cryptococcus might tune its cell surface to induce but survive repeated phagocytosis during early pathogenesis in the lung.

The anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of primary brain tumors and brain metastases. Phenothiazines (PTZs) have demonstrated anti-cancer properties through a variety of mechanisms. However, it remains unclear whether these effects are entirely separate from their activity as dopamine and serotonin receptor (DR/5-HTR) antagonists. Methods: In this study, we evaluated the anti-cancer efficacy of a novel PTZ analog, CWHM-974, that was shown to be 100-1000-fold less potent against DR/5-HTR than its analog fluphenazine (FLU). Results: CWHM-974 was more potent than FLU against a panel of cancer cell lines, thus clearly demonstrating that its anti-cancer effects were independent of DR/5-HTR signaling. Our results further suggested that calmodulin (CaM) binding may be necessary, but not sufficient, to explain the anti-cancer effects of CWHM-974. While both FLU and CWHM-974 induced apoptosis, they induced distinct effects on the cell cycle (G0/G1 and mitotic arrest respectively) suggesting that they may have differential effects on CaM-binding proteins involved in cell cycle regulation. Discussion: Altogether, our findings indicated that the anti-cancer efficacy of the CWHM-974 is separable from DR/5-HTR antagonism. Thus, reducing the toxicity associated with phenothiazines related to DR/5-HTR antagonism may improve the potential to repurpose this class of drugs to treat brain tumors and/or brain metastasis.

Cryptococcus neoformans adapts to the host environment through TOR-mediated remodeling of phospholipid asymmetry.

Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO2 concentrations are 100-fold higher than the external environment and strains unable to grow at host CO2 concentrations are not pathogenic. Using a genetic screening and transcriptional profiling approach, we report that the TOR pathway is critical for C. neoformans adaptation to host CO2 partly through Ypk1-dependent remodeling of phosphatidylserine asymmetry at the plasma membrane. We also describe a C. neoformans ABC/PDR transporter (PDR9) that is highly expressed in CO2-sensitive environmental strains, suppresses CO2-induced phosphatidylserine/phospholipid remodeling, and increases susceptibility to host concentrations of CO2. Interestingly, regulation of plasma membrane lipid asymmetry by the TOR-Ypk1 axis is distinct in C. neoformans compared to S. cerevisiae. Finally, host CO2 concentrations suppress the C. neoformans pathways that respond to host temperature (Mpk1) and pH (Rim101), indicating that host adaptation requires a stringent balance among distinct stress responses.

Carbon Dioxide Potentiates Flucytosine Susceptibility in Cryptococcus neoformans.

Cryptococcal meningoencephalitis remains a global health threat with limited treatment options. Currently, the most effective treatment regimens are based on a combination therapy of flucytosine with either amphotericin B or fluconazole. Slow but steady progress is being made toward universal access to flucytosine-based therapies. The broadening access to flucytosine combination therapies will be accompanied by the need for microbiological methods that reliably determine strain susceptibility. This is especially true considering that flucytosine susceptibility can vary widely across clinical isolates. Identifying culture conditions that best represent the host environment are likely optimal and may even be required for accurately determining in vivo flucytosine susceptibility. Here, we report that culture conditions incorporating host-like concentrations of carbon dioxide (CO2) potentiated flucytosine susceptibilities across clinical isolates (10 of 11) that exhibited a range of MIC values under ambient growth conditions (2 to 8 µg/mL) by standard Clinical and Laboratory Standards Institute susceptibility testing. CO2 induced a dose-dependent increase in flucytosine susceptibility between 2- and 8-fold over standard conditions. The CO2-dependent increase in flucytosine susceptibility did not correspond to an increase in fluorouracil susceptibility, indicating a central role for flucytosine uptake through the cytosine permease in the presence of host-like CO2 concentrations. Indeed, the expression of the cytosine permease gene (FCY2) was induced 18- to 60-fold in the mouse lung environment. Therefore, the activity of flucytosine is likely to be very dependent upon host environment and may not be well represented by standard in vitro susceptibility testing. IMPORTANCE Cryptococcus neoformans causes life-threatening infections of the brain. The most effective treatment regimens are based on flucytosine-based combination therapy, which has led to increasingly successful broadening of access to flucytosine globally. Wider use of flucytosine-based therapies for cryptococcal infections will require the ability to reliably determine clinical isolate susceptibilities. We showed that host-like carbon dioxide stress affected flucytosine susceptibility, and this likely occurred through flucytosine uptake. We further showed that the gene encoding the permease, FCY2, and that is responsible for flucytosine uptake was strongly induced during cryptococcal infection. Our data provide insights into the distinctions between the activity of flucytosine in the host environment and during in vitro susceptibility testing.

GAPDH mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites.

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Identifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH in MEP pathway control and antimalarial sensitivity.

Genetic Interaction Analysis Reveals that Cryptococcus neoformans Utilizes Multiple Acetyl-CoA-Generating Pathways during Infection.

Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. Previous work has shown that two nonessential acetyl-CoA metabolism enzymes, ATP-citrate lyase (ACL1) and acetyl-CoA synthetase (ACS1), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1Δ kbc1Δ double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log10 CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1Δ mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment. IMPORTANCE The adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of nonessential genes (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.

FKS1 Is Required for Cryptococcus neoformans Fitness In Vivo: Application of Copper-Regulated Gene Expression to Mouse Models of Cryptococcosis.

There is an urgent need for new antifungals to treat cryptococcal meningoencephalitis, a leading cause of mortality in people living with HIV/AIDS. An important aspect of antifungal drug development is the validation of targets to determine whether they are required for the survival of the organism in animal models of disease. In Cryptococcus neoformans, a copper-regulated promoter (pCTR4-2) has been used previously to modulate gene expression in vivo. The premise for these experiments is that copper concentrations differ depending on the host niche. Here, we directly test this premise and confirm that the expression of CTR4, the promoter used to regulate gene expression, is much lower in the mouse lung compared to the brain. To further explore this approach, we applied it to the gene encoding 1,3-ß-glucan synthase, FKS1. In vitro, reduced expression of FKS1 has little effect on growth but does activate the cell wall integrity stress response and increase susceptibility to caspofungin, a direct inhibitor of Fks1. These data suggest that compensatory pathways that reduce C. neoformans resistance do so through posttranscriptional effects. In vivo, however, a less pronounced reduction in FKS1 expression leads to a much more significant reduction in lung fungal burden (~1 log10 CFU), indicating that the compensatory responses to a reduction in FKS1 expression are not as effective in vivo as they are in vitro. In summary, use of copper-regulated expression of putative drug targets in vitro and in vivo can provide insights into the biological consequences of reduced activity of the target during infection. IMPORTANCE Conditional expression systems are widely used to genetically validate antifungal drug targets in mouse models of infection. Copper-regulated expression using the promoter of the CTR4 gene has been sporadically used for this purpose in C. neoformans. Here, we show that CTR4 expression is low in the lung and high in the brain, establishing the basic premise behind this approach. We applied the approach to the study of FKS1, the gene encoding the target of the echinocandin class of 1,3-ß-glucan synthase inhibitors. Our in vitro and in vivo studies indicate that C. neoformans tolerates extremely low levels of FKS1 expression. This observation provides a potential explanation for the poor activity of 1,3-ß-glucan synthase inhibitors toward C. neoformans.

Gluconate Kinase Is Required for Gluconate Assimilation and Sporulation in Cryptococcus neoformans.

Cryptococcus neoformans is an environmental yeast and an opportunistic human pathogen. The ability to cause disease depends on the ability to adapt to the human host. Previous studies implicated infectivity-related kinase 3 (IRK3, CNAG_03048) as required for establishing an infection. We genetically and biochemically characterized IRK3 as a gluconate kinase and propose the name GNK1. This metabolic enzyme utilizes gluconate to produce 6-phosphogluconate as part of the alternative oxidative phase of the pentose phosphate pathway (AOXPPP). The presence of GNK1 confirms that the AOXPPP is present and able to compensate for loss of the traditional OXPPP, providing an explanation for its nonessentiality. C. neoformans can utilize gluconate as an alternative carbon source in a GNK1-dependent manner. In our efforts to understand the role of GNK1 in host adaptation and virulence, we found that GNK1-deficient mutants have variable virulence and carbon dioxide tolerance across multiple strains, suggesting that second site mutations frequently interact with GNK1 deletion mutations. In our effort to isolate these genetic loci by backcrossing experiments, we discovered that GNK1-deficient strains are unable to sporulate. These data suggest that gluconate metabolism is critical for sporulation of C. neoformans. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that contributes to nearly 180,000 deaths annually. We characterized a gene named GNK1 that appears to interact with other genetic loci involved with the ability of C. neoformans to act as a pathogen. While these interacting genetic loci remain elusive, we discovered that GNK1 plays roles in both metabolism and mating/sporulation. Further interrogation of the mechanistic role for GNK1 in sexual reproduction may uncover a larger network of genes that are important for host adaptation and virulence.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence.

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

Targeting Host Glycolysis as a Strategy for Antimalarial Development.

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase - a critical enzyme in glycolysis - and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

Protein Prenylation and Hsp40 in Thermotolerance of Plasmodium falciparum Malaria Parasites.

During its complex life cycle, the malaria parasite survives dramatic environmental stresses, including large temperature shifts. Protein prenylation is required during asexual replication of Plasmodium falciparum, and the canonical heat shock protein 40 protein (HSP40; PF3D7_1437900) is posttranslationally modified with a 15-carbon farnesyl isoprenyl group. In other organisms, farnesylation of Hsp40 orthologs controls their localization and function in resisting environmental stress. In this work, we find that plastidial isopentenyl pyrophosphate (IPP) synthesis and protein farnesylation are required for malaria parasite survival after cold and heat shock. Furthermore, loss of HSP40 farnesylation alters its membrane attachment and interaction with proteins in essential pathways in the parasite. Together, this work reveals that farnesylation is essential for parasite survival during temperature stress. Farnesylation of HSP40 may promote thermotolerance by guiding distinct chaperone-client protein interactions.

Potent, specific MEPicides for treatment of zoonotic staphylococci.

Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, that DXR represents a promising, druggable target for future development.

Suppression of Drug Resistance Reveals a Genetic Mechanism of Metabolic Plasticity in Malaria Parasites.

In the malaria parasite Plasmodium falciparum, synthesis of isoprenoids from glycolytic intermediates is essential for survival. The antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In P. falciparum, we identified a loss-of-function mutation in HAD2 (P. falciparum 3D7_1226300 [PF3D7_1226300]) as necessary for FSM resistance. Enzymatic characterization revealed that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, is a phosphatase. Harnessing a growth defect in resistant parasites, we selected for suppression of HAD2-mediated FSM resistance and uncovered hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase 9 (PFK9). Metabolic profiling demonstrated that FSM resistance is achieved via increased steady-state levels of methylerythritol phosphate (MEP) pathway and glycolytic intermediates and confirmed reduced PFK9 function in the suppressed strains. We identified HAD2 as a novel regulator of malaria parasite metabolism and drug sensitivity and uncovered PFK9 as a novel site of genetic metabolic plasticity in the parasite. Our report informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveals a previously undescribed strategy by which malaria parasites adapt to cellular metabolic dysregulation.IMPORTANCE Unique and essential aspects of parasite metabolism are excellent targets for development of new antimalarials. An improved understanding of parasite metabolism and drug resistance mechanisms is urgently needed. The antibiotic fosmidomycin targets the synthesis of essential isoprenoid compounds from glucose and is a candidate for antimalarial development. Our report identifies a novel mechanism of drug resistance and further describes a family of metabolic regulators in the parasite. Using a novel forward genetic approach, we also uncovered mutations that suppress drug resistance in the glycolytic enzyme PFK9. Thus, we identify an unexpected genetic mechanism of adaptation to metabolic insult that influences parasite fitness and tolerance of antimalarials.

De novo Assembly and Analysis of the Northern Leopard Frog Rana pipiens Transcriptome.

The northern leopard frog Rana (Lithobates) pipiens is an important animal model, being used extensively in cancer, neurology, physiology, and biomechanical studies. R. pipiens is a native North American frog whose range extends from northern Canada to southwest United States, but over the past few decades its populations have declined significantly and is now considered uncommon in large portions of the United States and Canada. To aid in the study and conservation of R. pipiens, this paper describes the first R. pipiens transcriptome. The R. pipiens transcriptome was annotated using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Eukaryotic Orthologous Groups (KOG). Differential expression analysis revealed universal and tissue specific genes, and endocrine-related genes were identified. Transcriptome assemblies and other sequence data are available for download.

A novel method for simulating insulin mediated GLUT4 translocation.

Glucose transport in humans is a vital process which is tightly regulated by the endocrine system. Specifically, the insulin hormone triggers a cascade of intracellular signals in target cells mediating the uptake of glucose. Insulin signaling triggers cellular relocalization of the glucose transporter protein GLUT4 to the cell surface, which is primarily responsible for regulated glucose import. Pathology associated with the disruption of this pathway can lead to metabolic disorders, such as type II diabetes mellitus, characterized by the failure of cells to appropriately uptake glucose from the blood. We describe a novel simulation tool of the insulin intracellular response, incorporating the latest findings regarding As160 and GEF interactions. The simulation tool differs from previous computational approaches which employ algebraic or differential equations; instead, the tool incorporates statistical variations of kinetic constants and initial molecular concentrations which more accurately mimic the intracellular environment. Using this approach, we successfully recapitulate observed in vitro insulin responses, plus the effects of Wortmannin-like inhibition of the pathway. The developed tool provides insight into transient changes in molecule concentrations throughout the insulin signaling pathway, and may be employed to identify or evaluate potentially critical components of this pathway, including those associated with insulin resistance. In the future, this highly tractable platform may be useful for simulating other complex cell signaling pathways. Biotechnol. Bioeng. 2014;111: 2454-2465. © 2014 Wiley Periodicals, Inc.