Compatibility of Evolutionary Responses to Constituent Antibiotics Drive Resistance Evolution to Drug Pairs

Antibiotic combinations are considered a relevant strategy to tackle the global antibiotic resistance crisis since they are believed to increase treatment efficacy and reduce resistance evolution (WHO treatment guidelines for drug-resistant tuberculosis: 2016 update.). However, studies of the evolution of bacterial resistance to combination therapy have focused on a limited number of drugs and have provided contradictory results (Lipsitch, Levin BR. 1997; Hegreness et al. 2008; Munck et al. 2014). To address this gap in our understanding, we performed a large-scale laboratory evolution experiment, adapting eight replicate lineages of Escherichia coli to a diverse set of 22 different antibiotics and 33 antibiotic pairs. We found that combination therapy significantly limits the evolution of de novode novo resistance in E. coli, yet different drug combinations vary substantially in their propensity to select for resistance. In contrast to current theories, the phenotypic features of drug pairs are weak predictors of resistance evolution. Instead, the resistance evolution is driven by the relationship between the evolutionary trajectories that lead to resistance to a drug combination and those that lead to resistance to the component drugs. Drug combinations requiring a novel genetic response from target bacteria compared with the individual component drugs significantly reduce resistance evolution. These data support combination therapy as a treatment option to decelerate resistance evolution and provide a novel framework for selecting optimized drug combinations based on bacterial evolutionary responses.     

Synergistic inhibition of endothelial cell proliferation, tube formation, and sprouting by cyclosporin A and itraconazole

Pathological angiogenesis contributes to a number of diseases including cancer and macular degeneration. Although angiogenesis inhibitors are available in the clinic, their efficacy against most cancers is modest due in part to the existence of alternative and compensatory signaling pathways. Given that angiogenesis is dependent on multiple growth factors and a broad signaling network in vivo, we sought to explore the potential of multidrug cocktails for angiogenesis inhibition. We have screened 741 clinical drug combinations for the synergistic inhibition of endothelial cell proliferation. We focused specifically on existing clinical drugs since the re-purposing of clinical drugs allows for a more rapid and cost effective transition to clinical studies when compared to new drug entities. Our screen identified cyclosporin A (CsA), an immunosuppressant, and itraconazole, an antifungal drug, as a synergistic pair of inhibitors of endothelial cell proliferation. In combination, the IC(50) dose of each drug is reduced by 3 to 9 fold. We also tested the ability of the combination to inhibit endothelial cell tube formation and sprouting, which are dependent on two essential processes in angiogenesis, endothelial cell migration and differentiation. We found that CsA and itraconazole synergistically inhibit tube network size and sprout formation. Lastly, we tested the combination on human foreskin fibroblast viability as well as Jurkat T cell and HeLa cell proliferation, and found that endothelial cells are selectively targeted. Thus, it is possible to combine existing clinical drugs to synergistically inhibit in vitro models of angiogenesis. This strategy may be useful in pursuing the next generation of antiangiogenesis therapy.     

Hedging against Antiviral Resistance during the Next Influenza Pandemic Using Small Stockpiles of an Alternative Chemotherapy

Background

The effectiveness of single-drug antiviral interventions to reduce morbidity and mortality during the next influenza pandemic will be substantially weakened if transmissible strains emerge which are resistant to the stockpiled antiviral drugs. We developed a mathematical model to test the hypothesis that a small stockpile of a secondary antiviral drug could be used to mitigate the adverse consequences of the emergence of resistant strains.

Methods and Findings

We used a multistrain stochastic transmission model of influenza to show that the spread of antiviral resistance can be significantly reduced by deploying a small stockpile (1% population coverage) of a secondary drug during the early phase of local epidemics. We considered two strategies for the use of the secondary stockpile: early combination chemotherapy (ECC; individuals are treated with both drugs in combination while both are available); and sequential multidrug chemotherapy (SMC; individuals are treated only with the secondary drug until it is exhausted, then treated with the primary drug). We investigated all potentially important regions of unknown parameter space and found that both ECC and SMC reduced the cumulative attack rate (AR) and the resistant attack rate (RAR) unless the probability of emergence of resistance to the primary drug p_A was so low (less than 1 in 10,000) that resistance was unlikely to be a problem or so high (more than 1 in 20) that resistance emerged as soon as primary drug monotherapy began. For example, when the basic reproductive number was 1.8 and 40% of symptomatic individuals were treated with antivirals, AR and RAR were 67% and 38% under monotherapy if p _A = 0.01. If the probability of resistance emergence for the secondary drug was also 0.01, then SMC reduced AR and RAR to 57% and 2%. The effectiveness of ECC was similar if combination chemotherapy reduced the probabilities of resistance emergence by at least ten times. We extended our model using travel data between 105 large cities to investigate the robustness of these resistance-limiting strategies at a global scale. We found that as long as populations that were the main source of resistant strains employed these strategies (SMC or ECC), then those same strategies were also effective for populations far from the source even when some intermediate populations failed to control resistance. In essence, through the existence of many wild-type epidemics, the interconnectedness of the global network dampened the international spread of resistant strains.

Conclusions

Our results indicate that the augmentation of existing stockpiles of a single anti-influenza drug with smaller stockpiles of a second drug could be an effective and inexpensive epidemiological hedge against antiviral resistance if either SMC or ECC were used. Choosing between these strategies will require additional empirical studies. Specifically, the choice will depend on the safety of combination therapy and the synergistic effect of one antiviral in suppressing the emergence of resistance to the other antiviral when both are taken in combination.     

Application of drug metabolising mutants of cytochrome P450 BM3 (CYP102A1) as biocatalysts for the generation of reactive metabolites

Recently, several mutants of cytochrome P450 BM3 (CYP102A1) with high activity toward drugs have been obtained by a combination of site-directed and random mutagenesis. In the present study, the applicability of these mutants as biocatalysts in the production of reactive metabolites from the drugs clozapine, diclofenac and acetaminophen was investigated. We showed that the four CYP102A1 mutants used in this study formed the same metabolites as human and rat liver microsomes, with an activity up to 70-fold higher compared to human enzymes. Using these CYP102A1 mutants, three novels GSH adducts of diclofenac were discovered which were also formed in incubations with human liver microsomes. This work shows that CYP102A1 mutants are very useful tools for the generation of high levels of reference metabolites and reactive intermediates of drugs. Producing high levels of those reactive metabolites, that might play a role in adverse drug reactions (ADRs) in humans, will facilitate their isolation, structural elucidation, and could be very useful for the toxicological characterization of novel drugs and/or drug candidates.     

Delaying antimalarial drug resistance with combination chemotherapy

Resistance to antimalarial drugs arises when spontaneously occurring mutants with gene mutations or amplifications which confer reduced drug susceptibility are selected, and are then transmitted. Simultaneous use of two or more antimalarials with different modes of action and which therefore do not share the same resistance mechanisms will reduce the chance of selection, because the chance of a resistant mutant surviving is the product of the parasite mutation rates for the individual drugs, multiplied by the number of parasites in an infection that are exposed to the drugs. The artemisinin derivatives are very active antimalarials, which produce large reductions in parasite biomass per asexual cycle, and reduce malaria transmissibility. To date no resistance to these drugs has been reported. These drugs therefore make particularly effective combination partners. This suggests that antimalarial drugs should not be used alone in treatment, but always in combination, as in the treatment of tuberculosis or HIV, and that the combination should include artemisinin or one of its derivatives.     

Efficiency of antisense oligonucleotide drug discovery

The costs for discovering and developing new drugs continue to escalate, with current estimates that the average cost is more than $800 million for each new drug brought to the market. Pharmaceutical companies are under enormous pressure to increase their efficiency for bringing new drugs to the market by third-party payers, shareholders, and their patients, and at the same time regulators are placing increased demands on the industry. To be successful in the future, pharmaceutical companies must change how they discover and develop new drugs. So far, new technologies have done little to increase overall efficiency of the industry and have added additional costs. Platform technologies such as monoclonal antibodies and antisense oligonucleotides have the potential of reducing costs for discovery of new drugs, in that many of the steps required for traditional small molecules can be skipped or streamlined. Additionally the success of identifying a drug candidate is much higher with platform technologies compared to small molecule drugs. This review will highlight some of the efficiencies of antisense oligonucleotide drug discovery compared to traditional drugs and will point out some of the current limitations of the technology.     

Benznidazole/Itraconazole Combination Treatment Enhances Anti-Trypanosoma cruzi Activity in Experimental Chagas Disease

The nitroheterocyclic drugs nifurtimox and benznidazole are first-line drugs available to treat Chagas disease; however, they have limitations, including long treatment courses and toxicity. Strategies to overcome these limitations include the identification of new drugs with specific target profiles, re-dosing regimens for the current drugs, drug repositioning and combination therapy. In this work, we evaluated combination therapy as an approach for optimization of the current therapeutic regimen for Chagas disease. The curative action of benznidazole/itraconazole combinations was explored in an established infection of the mice model with the T. cruzi Y strain. The activities of the benznidazole/itraconazole combinations were compared with the results from those receiving the same dosage of each individual drug. The administration of benznidazole/itraconazole in combination eliminated parasites from the blood more efficiently than each drug alone. Here, there was a significant reduction of the number of treatment days (number of doses) necessary to induce parasitemia suppression with the benznidazole/itraconazole combination, as compared to each compound administered alone. These results clearly indicate the enhanced effects of these drugs in combination, particularly at the dose of 75 mg/kg, as the effects observed with the drug combinations were four times more effective than those of each drug used alone. Moreover, benznidazole/itraconazole treatment was shown to prevent or decrease the typical lesions associated with chronic experimental Chagas disease, as illustrated by similar levels of inflammatory cells and fibrosis in the cardiac muscle tissue of healthy and treated mice. These results emphasize the importance of exploring the potential of combination treatments with currently available compounds to specifically treat Chagas disease.     

Variability in Response to Drugs

Variability in the response to drugs is due to three principal components—the disease, the responsiveness of tissues, and the concentration of the drug at its site of action (as reflected by its plasma concentration). The relative contributions of these components will differ not only for different drugs but also for different effects of the same drug. Rational drug therapy depends on knowledge of all three factors.     

Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a “search and capture” behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.     

Synergistic Inhibition by Benzodiazepine and Barbiturate Drugs of the Clock Control of Ovulation in Hamsters

A surge of pituitary luteinizing hormone (LH) into the bloodstream occurs in hamsters every 4 days between 1:30 p.m. and 3 p.m. in response to a signal from a biological clock. This surge initiates behavioral estrus ∼2 h later and ovulation ∼12 h later. Phenobarbital at a dose ≥100 mg/kg consistently blocks LH release. Barbiturate and benzodiazepine drugs have separate binding sites in the GABA_A receptor/chloride channel complex. Binding of either drug increases GABA-mediated chloride conductance, which suppresses the postsynaptic neuron. Barbiturate binding also increases benzodiazepine binding. This suggested that these drugs might synergize to inhibit LH release. A combination of triazolam and phenobarbital at doses of 10 mg/kg injected s.c. at 1:30 p.m. inhibited ovulation and extended the 4-day vaginal cycle in all treated hamsters. Either drug dose injected alone at 1:30 p.m., or the combination at 3 p.m., was completely ineffective. Bicuculline prevented inhibition by the combination at 1:30 p.m. The clock signal for LH release may act by antagonizing GABA transmission, which may be chronically inhibiting LH release. The combination delimited a 75-min period (1:30-2:45 p.m.) within which the clock signal for LH release occurred in all individuals (ET₅₀ = 2:08 p.m.). This period appears to arise from individuals with different but constant clock settings rather than from a 75-min variation in the clock setting of the individual.     

STRN-ALK rearranged pediatric malignant peritoneal mesothelioma – Functional testing of 527 cancer drugs in patient-derived cancer cells

Genetic rearrangements involving the anaplastic lymphoma kinase (ALK) gene create oncogenic drivers for several cancers, including malignant peritoneal mesothelioma (MPeM). Here, we report genomic and functional precision oncology profiling on a rare case of a 5-year old patient diagnosed with wide-spread and aggressive MPeM, driven by STRN-ALK rearrangement. We established genomically representative patient-derived cancer cells (PDCs) from the tumor sample and performed high-throughput drug sensitivity testing with 527 oncology compounds to identify potent inhibitors. As expected, the PDCs were overall sensitive to the ALK inhibitors, although the eight different inhibitors tested had variable efficacy. We also discovered other effective inhibitors, such as MEK/ERK inhibitors and those targeting pathways downstream of ALK as well as Bcl-xl inhibitors. In contrast, most cytotoxic drugs were not very effective. ALK inhibitors synergized with MEK and PI3K/mTOR inhibitors, highlighting potential combinatorial strategies to enhance drug efficacy and tackle drug resistance. Based on genomic data and associated functional validation, the patient was treated with the ALK inhibitor crizotinib in combination with conventional chemotherapy (cisplatin and gemcitabine). A complete disease remission was reached, lasting now for over 3 years. Our results illustrate a rare pediatric cancer case, and highlight the potential of functional precision oncology to discover pathogenetic drivers, validate dependency on driver signals, compare different inhibitors against each other and potentially enhance targeted treatments by drug combinations. Such real-time implementation of functional precision oncology could pave the way towards safer and more effective personalized cancer therapies for individual pediatric cancer patients with rare tumors.     

Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects

While it is currently estimated that 40 to 50% of eukaryotic proteins are phosphorylated, little is known about the frequency and local effects of phosphorylation near pharmaceutical inhibitor binding sites. In this study, we investigated how frequently phosphorylation may affect the binding of drug inhibitors to target proteins. We examined the 453 non‐redundant structures of soluble mammalian drug target proteins bound to inhibitors currently available in the Protein Data Bank (PDB). We cross‐referenced these structures with phosphorylation data available from the PhosphoSitePlus database. Three hundred twenty‐two of 453 (71%) of drug targets have evidence of phosphorylation that has been validated by multiple methods or labs. For 132 of 453 (29%) of those, the phosphorylation site is within 12 Å of the small molecule‐binding site, where it would likely alter small molecule binding affinity. We propose a framework for distinguishing between drug‐phosphorylation site interactions that are likely to alter the efficacy of drugs versus those that are not. In addition we highlight examples of well‐established drug targets, such as estrogen receptor alpha, for which phosphorylation may affect drug affinity and clinical efficacy. Our data suggest that phosphorylation may affect drug binding and efficacy for a significant fraction of drug target proteins. Proteins 2015; 83:25–36. © 2014 Wiley Periodicals, Inc.     

Benznidazole and Posaconazole in Experimental Chagas Disease: Positive Interaction in Concomitant and Sequential Treatments

Background

Current chemotherapy for Chagas disease is unsatisfactory due to its limited efficacy, particularly in the chronic phase, with frequent side effects that can lead to treatment discontinuation. Combined therapy is envisioned as an ideal approach since it may improve treatment efficacy whilst decreasing toxicity and the likelihood of resistance development. We evaluated the efficacy of posaconazole in combination with benznidazole on Trypanosoma cruzi infection in vivo.

Methods and Findings

Benznidazole and posaconazole were administered individually or in combination in an experimental acute murine infection model. Using a rapid treatment protocol for 7 days, the combined treatments were more efficacious in reducing parasitemia levels than the drugs given alone, with the effects most evident in combinations of sub-optimal doses of the drugs. Subsequently, the curative action of these drug combinations was investigated, using the same infection model and 25, 50, 75 or 100 mg/kg/day (mpk) of benznidazole in combination with 5, 10 or 20 mpk of posaconazole, given alone or concomitantly for 20 days. The effects of the combination treatments on parasitological cures were higher than the sum of such effects when the drugs were administered separately at the same doses, indicating synergistic activity. Finally, sequential therapy experiments were carried out with benznidazole or posaconazole over a short interval (10 days), followed by the second drug administered for the same period of time. It was found that the sequence of benznidazole (100 mpk) followed by posaconazole (20 mpk) provided cure rates comparable to those obtained with the full (20 days) treatments with either drug alone, and no cure was observed for the short treatments with drugs given alone.

Conclusions

Our data demonstrate the importance of investigating the potential beneficial effects of combination treatments with marketed compounds, and showed that combinations of benznidazole with posaconazole have a positive interaction in murine models of Chagas disease.     

华蟾素联合顺铂对人骨肉瘤U2OS细胞增殖和凋亡的影响

目的:研究华蟾素联合顺铂对人骨肉瘤U2OS细胞增殖和凋亡的影响,并探寻联合治疗增敏的可能分子机制。方法:采用不同浓度的华蟾素、顺铂单药和华蟾素联合顺铂处理人骨肉瘤U2OS细胞1-3天,通过CCK-8法检测其对U2OS细胞生长的抑制作用;平板克隆实验检测其对U2OS细胞集落形成能力的影响;流式细胞仪检测其对U2OS细胞凋亡的影响;RT-PCR及Western blotting检测Bax、Bcl-2、caspase-3、caspase-9等凋亡相关分子mRNA及蛋白水平的表达。结果:单用华蟾素或顺铂均可以浓度和时间依赖的方式抑制骨肉瘤U2OS细胞的增殖并诱导其凋亡,两药联合应用具有协同效应,并可以上调促凋亡基因Bax下调抑制凋亡基因Bcl-2的表达;联合用药组凋亡相关蛋白caspase-3、caspase-9的表达较单药组明显增加。结论:华蟾素联合顺铂与单一用药相比能够显著抑制人骨肉瘤细胞U2OS细胞的增殖,促进其凋亡,两药联合应用对骨肉瘤细胞的杀伤效应具有一定的协同效应,其机制可能与激活凋亡通路有关。     

Synergistic Interaction between Selective Drugs in Cell Populations Models

The design of selective drugs and combinatorial drug treatments are two of the main focuses in modern pharmacology. In this study we use a mathematical model of chimeric ligand-receptor interaction to show that the combination of selective drugs is synergistic in nature, providing a way to gain optimal selective potential at reduced doses compared to the same drugs when applied individually. We use a cell population model of proliferating cells expressing two different amounts of a target protein to show that both selectivity and synergism are robust against variability and heritability in the cell population. The reduction in the total drug administered due to the synergistic performance of the selective drugs can potentially result in reduced toxicity and off-target interactions, providing a mechanism to improve the treatment of cell-based diseases caused by aberrant gene overexpression, such as cancer and diabetes.     

Optimizing combination chemotherapy by controlling drug ratios

Cancer chemotherapy treatments typically employ drug combinations in which the dose of each agent is pushed to the brink of unacceptable toxicity; however, emerging evidence indicates that this approach may not be providing optimal efficacy due to the manner in which drugs interact. Specifically, whereas certain ratios of combined drugs can be synergistic, other ratios of the same agents may be antagonistic, implying that the most efficacious combinations may be those that utilize certain agents at reduced doses. Advances in nano-scale drug delivery vehicles now enable the translation of in vitro information on synergistic drug ratios into improved anticancer combination therapies in which the desired drug ratio can be controlled and maintained following administration in vivo, so that synergistic effects can be exploited. This "ratiometric" approach to combination chemotherapy opens new opportunities to enhance the effectiveness of existing and future treatment regimens across a spectrum of human diseases.     

Combined antimalarial therapy using artemisinin

The existing armamentarium of drugs for the treatment and prevention of malaria is limited primarily by resistance (and cross-resistance between closely related drugs). However, most of these drugs still have a place and their life-span could be prolonged if better deployed and used, and also by rationally combining them based on pharmacodynamic and pharmacokinetic properties. Newer compounds are also being developed. The nature of malaria disease and its prevalence in the developing world call for innovative approaches to develop new affordable drugs and to safeguard the available ones. According to WHO, the concept of combination therapy is based on the synergistic or additive potential of two or more drugs, to improve therapeutic efficacy and also delay the development of resistance to the individual components of the combination. Combination therapy (CT) with antimalarial drugs is the simultaneous use of two or more blood schizontocidal drugs with independent modes of action and different biochemical targets in the parasite. In the context of this definition, multiple-drug therapies that include a nonantimalarial drug to enhance the antimalarial effect of a blood schizontocidal drug are not considered combination therapy. Similarly, certain antimalarial drugs that fit the criteria of synergistic fixed-dose combinations are operationally considered as single products in that neither of the individual components would be given alone for anti-malarial therapy. An example is sulfadoxine-pyrimethamine. Artemisinin-based combination therapies have been shown to improve treatment efficacy and also contain drug resistance in South-East Asia. However, major challenges exist in the deployment and use of antimalarial drug combination therapies, particularly in Africa. These include: 1) the choice of drug combinations best suited for the different epidemiological situations; 2) the cost of combination therapy; 3) the timing of the introduction of combination therapy; 4) the operational obstacles to implementation, especially compliance. As a response to increasing levels of antimalarial resistance, the World Health Organization (WHO) recommends that all countries experiencing resistance to conventional monotherapies, such as chloroquine, amodiaquine or sulfadoxine/pyrimethamine, should use combination therapies, preferably those containing artemisinin derivatives (ACTs--artemisinin-based combination therapies) for malaria caused by Plasmodium falciparum. There is a promising role of such compounds in replacing or complementing current options. Since 1979, several different formulations of artemisinin and its derivatives have been produced and studied in China in several thousand patients for either P. falciparum or P. vivax malaria. To date, there is no evidence of drug resistance to these compounds. The use of artemisinin, artemether, arteether and artesunate for either uncomplicated or severe malaria is now spreading through almost all malarious areas of the world, although some of they have no patent protection, their development (with few exceptions) has not followed yet full international standards. Both artesunate, artemether and arteether are rapidly and extensively converted to their common bioactive metabolite, dihydroarte-misinin. WHO currently recommends the following therapeutic options: 1) artemether/lumefantrine; 2) artesunate plus amodiaquine; 3) artesunate plus sulfadoxine/pyrimethamine (in areas where SP efficacy remains high); 4) artesunate plus mefloquine (in areas with low to moderate transmission); and 5) amodiaquine plus sulfadoxine/pyrimethamine, in areas where efficacy of both amodiaquine and sulfadoxine/pyrimethamine remains high (mainly limited to countries in West Africa). This non artemisinin-based combination therapy is reserved as an interim option for countries, which, for whatever reason, are unable immediately to move to ACTs.     

A systems biology approach to identify effective cocktail drugs

Background

Complex diseases, such as Type 2 Diabetes, are generally caused by multiple factors, which hamper effective drug discovery. To combat these diseases, combination regimens or combination drugs provide an alternative way, and are becoming the standard of treatment for complex diseases. However, most of existing combination drugs are developed based on clinical experience or test-and-trial strategy, which are not only time consuming but also expensive.

Results

In this paper, we presented a novel network-based systems biology approach to identify effective drug combinations by exploiting high throughput data. We assumed that a subnetwork or pathway will be affected in the networked cellular system after a drug is administrated. Therefore, the affected subnetwork can be used to assess the drug's overall effect, and thereby help to identify effective drug combinations by comparing the subnetworks affected by individual drugs with that by the combination drug. In this work, we first constructed a molecular interaction network by integrating protein interactions, protein-DNA interactions, and signaling pathways. A new model was then developed to detect subnetworks affected by drugs. Furthermore, we proposed a new score to evaluate the overall effect of one drug by taking into account both efficacy and side-effects. As a pilot study we applied the proposed method to identify effective combinations of drugs used to treat Type 2 Diabetes. Our method detected the combination of Metformin and Rosiglitazone, which is actually Avandamet, a drug that has been successfully used to treat Type 2 Diabetes.

Conclusions

The results on real biological data demonstrate the effectiveness and efficiency of the proposed method, which can not only detect effective cocktail combination of drugs in an accurate manner but also significantly reduce expensive and tedious trial-and-error experiments.

     

Selective Targeting of CTNNB1-, KRAS- or MYC-Driven Cell Growth by Combinations of Existing Drugs

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for β-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes.     

基于小分子化合物库筛选协同多腺苷二磷酸核糖聚合酶抑制剂杀伤卵巢癌细胞的药物

多腺苷二磷酸核糖聚合酶(poly(ADP-ribose) polymerase, PARP)抑制剂是一类靶向 DNA 修复缺陷癌细胞的新型药物。早期研究表明 PARP 抑制剂取得了令人满意的结果,然而药物治疗后出现的耐药机制尚未完全揭露。因此,有必要寻找更多的靶向药物与PARP 抑制剂联用,以达到杀伤肿瘤细胞的目的。本文基于379种小分子化合物和PARP抑制剂尼拉帕尼(Niraparib)的联合用药筛选,通过细胞增殖实验、克隆存活实验和免疫荧光染色等方法筛选潜在的具有协同PARP抑制剂杀伤卵巢癌细胞的药物。结果表明,其中有8种小分子化合物具有较好的联合用药效果,包括2种已经报道的与PARP抑制剂具有联用效果的小分子化合物STF-118804和Disulfiram。我们从中选取原肌球蛋白受体激酶 A (tropomyosin receptor kinase A,TrKA)的抑制剂GW441756,进行了多种肿瘤细胞的验证以及初步机制的探究。Niraparib和TrKA抑制剂的联合用药显著增加肿瘤细胞对PARP抑制剂的敏感性(P<0.05)。从机制上分析,联合用药组细胞内γH2AX foci的数目显著增加(P<0.05),说明TrKA抑制剂阻碍损伤后细胞的DNA损伤修复能力;同时,联合用药显著降低细胞内同源重组修复(homologous recombination repair,HRR)标志物RAD51 foci(P<0.05)的形成,说明TrKA抑制剂可能通过抑制细胞的HRR效率阻碍细胞的DNA损伤修复。本研究的结果提示,TrKA抑制剂可以作为一种与PARP抑制剂联用杀伤卵巢癌细胞的潜在药物。