In Vitro Interaction of Geldanamycin with Triazoles and Echinocandins Against Common and Emerging Candida Species
Abstract
The escalating challenge of invasive fungal infections, particularly those caused by *Candida* species, continues to pose a significant threat to global public health, leading to substantial morbidity and mortality, especially in immunocompromised individuals. A major impediment to effective treatment lies in the increasing prevalence of antifungal drug resistance, which diminishes the efficacy of current therapeutic agents and necessitates the exploration of novel strategies. Heat shock protein 90 (Hsp90) is a crucial molecular chaperone that plays an indispensable role in the folding, stability, and function of numerous client proteins, many of which are vital for fungal virulence, stress adaptation, and drug resistance. Given its central role, Hsp90 has emerged as a promising target for antifungal drug development, particularly in combination therapies aimed at circumventing or overcoming established resistance mechanisms. This study was meticulously designed with the primary objective of comprehensively determining the *in vitro* interactive effects of geldanamycin, a well-characterized inhibitor of Hsp90, when combined with commonly used antifungal agents belonging to the triazole and echinocandin classes, against a panel of both prevalent and emerging *Candida* species.
To ensure clinical relevance and a broad representation of fungal pathogenicity and resistance profiles, our investigation included a selection of twenty clinically important *Candida* strains. This cohort comprised five distinct strains each of *Candida auris*, an emerging multidrug-resistant pathogen of significant global concern; *Candida albicans*, the most common and widely studied *Candida* species; *Candida parapsilosis*, frequently associated with catheter-related bloodstream infections; and *Candida glabrata*, another prevalent species known for its intrinsic and rapidly developing resistance to azole antifungals. The meticulous selection of these species allowed for a comprehensive assessment of the combination effects across varied genetic backgrounds and resistance patterns.
The *in vitro* interactions between geldanamycin and the selected antifungal drugs—specifically fluconazole and itraconazole (representing the triazole class), and caspofungin and anidulafungin (representing the echinocandin class)—were rigorously determined using the established checkerboard microdilution method. This quantitative technique allows for the precise evaluation of drug combinations by testing various concentrations of each agent in a matrix format. The results of these interaction studies were subsequently interpreted based on the fractional inhibitory concentration index (FICI) values. FICI is a widely accepted mathematical model that objectively classifies drug interactions as either synergistic (where the combined effect is greater than the sum of individual effects), indifferent (where the combined effect is no different from the sum of individual effects), or antagonistic (where the combined effect is less than the sum of individual effects, indicating an undesirable counteraction).
Our detailed analysis of the *in vitro* combination of fluconazole with geldanamycin yielded highly encouraging results. A significant synergistic effect was consistently observed against all five tested strains of *Candida albicans* (100% synergy), along with a high proportion of strains from *Candida glabrata* (80% synergy) and *Candida parapsilosis* (80% synergy). The FICI values for these synergistic interactions ranged from 0.009 to 0.5, unequivocally confirming a potent collaborative effect. This synergy translated into a remarkable reduction in the overall minimum inhibitory concentration (MIC) range of fluconazole against these susceptible species. Specifically, the MIC range for fluconazole against *C. albicans, C. glabrata*, and *C. parapsilosis* plummeted from a relatively high range of 16-256 mg/L when tested as a single agent, to a significantly lower and more therapeutically achievable range of 0.25-64 mg/L when combined with geldanamycin. However, in stark contrast, the combination of fluconazole with geldanamycin resulted in consistently indifferent interactions against all strains of *Candida auris*, with FICI values ranging from 1.5 to 2. This suggests that this combination strategy is ineffective against this particular emerging pathogen.
Regarding the interaction of geldanamycin with itraconazole, another vital triazole antifungal, we observed an even more pronounced synergistic effect. This synergy was consistently evident against all tested strains of *Candida albicans, Candida glabrata*, and *Candida parapsilosis*. The FICI values for these combinations ranged from a very low 0.009 to 0.375, indicating a strong and reliable synergistic interaction. Concomitantly, the MIC range of itraconazole against these susceptible species was dramatically reduced from its single-agent range of 0.125-32 mg/L, to an exceptionally low and clinically promising range of 0.03-1 mg/L when combined with geldanamycin. This profound reduction highlights the potential of this combination to significantly enhance itraconazole’s antifungal potency and possibly overcome some levels of existing azole resistance in these species.
In assessing the interactions with the echinocandin class, combinations of geldanamycin with both caspofungin and anidulafungin yielded consistently indifferent effects across all the *Candida* species evaluated in this study, including *C. auris, C. albicans, C. parapsilosis*, and *C. glabrata*. This indifference suggests that simultaneously targeting Hsp90 and the fungal cell wall (the target of echinocandins) does not result in a synergistic or antagonistic interaction, implying distinct or non-overlapping mechanisms of action for these specific drug pairs. Importantly, throughout the entire comprehensive study, no instances of antagonism were observed in any of the tested combinations, which is a critical positive finding for the potential clinical applicability of such combination strategies.
In conclusion, our findings strongly suggest that the simultaneous targeting of Hsp90 with geldanamycin, coupled with the inhibition of lanosterol 14-α demethylase (the molecular target of azole antifungals like fluconazole and itraconazole), represents a highly effective and promising therapeutic approach against common and clinically significant *Candida* species, specifically *Candida albicans, Candida glabrata*, and *Candida parapsilosis*. This combination strategy significantly enhances the antifungal activity of azoles, primarily through a marked reduction in their minimum inhibitory concentrations, implying potential for improved efficacy and reduced toxicity in clinical settings. However, it is crucial to note that this specific combination strategy proved ineffective against the emerging and notoriously resistant pathogen, *Candida auris*, underscoring the unique challenges posed by this species and the necessity for continued research into alternative therapeutic strategies for its management. The results pave the way for further investigation into the clinical utility of Hsp90 inhibitors in combination with azoles for the treatment of susceptible candidiasis.
Introduction
Invasive *Candida* infections, particularly those manifesting as bloodstream infections (candidemia), represent a profoundly life-threatening clinical challenge, carrying an alarmingly high overall mortality rate that ranges from 36% to a staggering 63%, depending on the specific patient population and underlying comorbidities. Historically, *Candida albicans* has been the predominant causative agent responsible for the vast majority of these cases. However, in recent years, there has been a notable and concerning epidemiological shift, with a significant increase in the frequency of infections attributed to non-*albicans Candida* species. This evolving landscape of candidiasis poses new diagnostic and therapeutic dilemmas. Among these emerging pathogens, *Candida glabrata* is frequently reported as the second most common cause of candidemia globally and stands as the leading cause of invasive fungal infections in specific vulnerable patient groups, such as those suffering from hematological malignancies, where antifungal resistance is a particular concern. *Candida parapsilosis* constitutes another critically important non-*albicans Candida* species, accounting for a substantial proportion, specifically between 17% and 50%, of all fungemia cases observed among pediatric patients, often linked to indwelling medical devices. Compounding these challenges, *Candida auris* has emerged onto the global stage as one of the top ten most serious and urgent fungal pathogens. This highly problematic species is notorious for causing large-scale hospital outbreaks and is characterized by a pervasive multidrug-resistant phenotype, rendering many conventional antifungal agents ineffective and severely limiting treatment options. Despite the steadily growing prevalence and escalating severity of *Candida* infections, the repertoire of antifungal drugs currently available for clinical treatment remains regrettably limited. Furthermore, the insidious development of drug resistance, whether intrinsic to certain species or acquired through prolonged drug exposure, has been widely reported across several *Candida* species, further diminishing the already constrained therapeutic arsenal. In light of these critical limitations and the urgent unmet medical need, the exploration of alternative therapeutic strategies, particularly innovative combination approaches, has become indispensable.
Drug combination therapy presents a highly promising and multifaceted method to overcome the aforementioned challenges. This strategy holds the potential to significantly improve the overall efficacy of existing drugs, potentially allowing for lower individual drug concentrations and reducing associated toxicities. Crucially, it may also help to decrease the rate at which resistant phenotypes develop, by simultaneously targeting multiple pathways or by synergistically impairing fungal survival. Moreover, in some instances, combination therapy has even demonstrated the remarkable ability to reverse established drug resistance, restoring the susceptibility of otherwise refractory pathogens. A key molecular target that has attracted considerable attention in this context is Heat shock protein 90 (Hsp90). Hsp90 is a vital and ubiquitous molecular chaperone that acts as a crucial regulator of the function, stability, and proper folding of a diverse array of client proteins within the cell. In *Candida* species, Hsp90 has been unequivocally shown to contribute significantly to various aspects of fungal biology critical for pathogenicity, including virulence expression, robust biofilm formation, intricate morphogenesis, and, importantly, the acquisition and maintenance of drug resistance, particularly against azole antifungals. Given its central and pleiotropic roles in fungal physiology and pathology, inhibitors of Hsp90 hold significant promise as antifungal agents, especially in combinatorial regimens. Promising *in vitro* activity of geldanamycin, a well-characterized Hsp90-inhibitor, has been reported when combined with various antifungal drugs against a range of filamentous fungi. Furthermore, specific antifungal activity of geldanamycin itself and, more pertinently, its synergistic interaction when combined with fluconazole against *C. albicans*, have been previously observed. However, despite these encouraging preliminary findings, there remains a significant dearth of knowledge regarding the activity of Hsp90-inhibitors, and specifically the combination of geldanamycin with newer generations of antifungal drugs, against not only common *Candida* species but also, and critically, against newly described and emerging pathogens such as *Candida auris*.
Therefore, the overarching aim of this meticulously designed study was to comprehensively evaluate the antifungal activity of geldanamycin as a standalone agent and, more importantly, to determine its interactive effects in combination with key antifungal drugs. These drugs included fluconazole and itraconazole, representing the widely used triazole class, and caspofungin and anidulafungin, representing the echinocandin class. Our investigation specifically targeted a panel of common *Candida* species (*C. albicans, C. glabrata, C. parapsilosis*) and, with particular emphasis, the highly concerning emerging pathogen *C. auris*, aiming to provide crucial insights into novel therapeutic strategies for candidiasis.
Materials and Methods
To ensure the clinical relevance and generalizability of our findings, a meticulously selected panel of twenty distinct *Candida* isolates, encompassing four different species, was utilized for this study. This panel included five isolates each of *Candida auris*, five of *Candida albicans*, five of *Candida parapsilosis*, and five of *Candida glabrata*. The precise identification of each isolate had been rigorously confirmed prior to this study through gold-standard molecular techniques, specifically sequencing of the D1/D2 region of ribosomal DNA and the internal transcribed spacer (ITS rDNA) region, ensuring accurate species assignment. Furthermore, based on the established guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI) M27-S4 guide for antifungal susceptibility testing, and considering the recommended tentative minimum inhibitory concentration (MIC) breakpoints for *C. auris* provided by the Centers for Disease Control and Prevention (CDC), all selected strains exhibited resistance to fluconazole, providing a challenging and clinically relevant context for evaluating combination therapies.
The *in vitro* interactions between geldanamycin, obtained from Cayman Chemical (Ann Arbor, Michigan, USA), and the chosen antifungal drugs were systematically determined. The antifungal drugs included fluconazole (Pfizer Central Research, Sandwich, UK), itraconazole (Janssen Research Foundation, Beerse, Belgium), caspofungin (Merck Sharp & Dohme, Haarlem, Netherlands), and anidulafungin (Pfizer Central Research, Sandwich, UK). The specific method employed for these interaction studies was the checkerboard microdilution assay, a well-established and highly quantitative technique for assessing synergistic or antagonistic drug combinations. To prepare the stock solutions, all drug powders were carefully dissolved in dimethyl sulfoxide (DMSO). Subsequent serial dilutions of each drug were meticulously prepared following the guidelines outlined in the CLSI M27 guide (4th edition), ensuring precise and reproducible concentrations. This process yielded final concentration ranges for the assay plates: 0.5–32 mg/L for geldanamycin, 0.25–128 mg/L for fluconazole, 0.03–16 mg/L for itraconazole, and 0.015–8 mg/L for both caspofungin and anidulafungin.
For the preparation of the 96-well test plates used in the checkerboard assay, a systematic dispensing strategy was followed. A volume of 50 µL of each serially diluted concentration of geldanamycin was dispensed into rows A through G, creating a gradient of geldanamycin concentrations across the rows. Similarly, 50 µL of each concentration of the antifungal drugs (fluconazole, itraconazole, caspofungin, or anidulafungin) was dispensed into columns 1 through 10, establishing a perpendicular gradient. Row H and column 11 were specifically designated to contain 100 µL of geldanamycin alone and the antifungal drugs alone, respectively, serving as single-drug controls for calculating individual MICs. Column 12 was reserved as a drug-free growth control, containing only medium and *Candida* suspension, to ensure optimal fungal growth in the absence of inhibitors. Suspensions of the *Candida* isolates were prepared according to the standardized method described in the CLSI M27-S4 standard, ensuring consistent inoculum sizes. Subsequently, 100 µL of the *Candida* suspension was added to each well of the test plates, initiating the culture.
The inoculated plates were then meticulously incubated at a controlled temperature of 35°C. After a 24-hour incubation period, the results were visually read to assess fungal growth inhibition. A reduction of 50% or more in fungal growth within a well, compared to the drug-free growth control wells, was considered significant inhibition. In instances where results were “off-scale” (meaning the MIC was higher than the highest tested concentration), these were conservatively shifted to the next higher concentration to facilitate FICI calculation. For the quantitative interpretation of the checkerboard assay results, the fractional inhibitory concentration index (FICI) was calculated using the following formula: FICI = (MIC drug A in combination / MIC drug A alone) + (MIC drug B in combination / MIC drug B alone). Based on established FICI criteria, interactions were categorized as synergism when FICI values were less than or equal to 0.5, indifference when FICI values ranged between 0.5 and 4 (inclusive of 0.5 but exclusive of 4), and antagonism when FICI values were greater than or equal to 4. To ensure the robustness and reproducibility of our experimental findings, all experiments were diligently performed in duplicate.
Results
Our comprehensive *in vitro* investigation revealed distinct patterns of interaction between geldanamycin and the various antifungal agents tested against the clinically relevant *Candida* species. The specific MIC values for fluconazole and itraconazole, both as single agents and in combination with geldanamycin, demonstrated notable shifts that informed our interpretation of drug interactions.
A highly encouraging synergistic effect for the combination of fluconazole and geldanamycin was consistently observed. This synergy was evident against all five tested strains of *Candida albicans* (100% of strains), a significant majority of *Candida glabrata* strains (80%), and a substantial proportion of *Candida parapsilosis* strains (80%). When combined with geldanamycin, the MIC ranges of fluconazole against these susceptible species were profoundly reduced, indicating a significant potentiation of its antifungal activity. Specifically, the MIC range for *C. albicans* decreased from 64–256 mg/L when fluconazole was used alone, to a remarkably lower range of 0.5 mg/L in combination. For *C. glabrata*, the MIC range reduced from 64–128 mg/L to 0.5–64 mg/L, and for *C. parapsilosis*, it decreased from 16–256 mg/L to 0.25–16 mg/L. These substantial reductions in MIC highlight the potential of this combination to overcome high levels of fluconazole resistance in these species, making otherwise resistant strains susceptible.
The synergistic effect was even more pronounced when itraconazole was combined with geldanamycin. This combination exhibited an excellent synergistic effect against all tested strains of *C. albicans*, *C. glabrata*, and *C. parapsilosis*, with FICI values consistently ranging from 0.009 to 0.375, signifying robust synergy. This led to an impressive change in the MIC range for itraconazole: against *C. albicans*, it dropped from a standalone range of 32 mg/L to an exceptionally low 0.03 mg/L; for *C. glabrata*, the range shifted from 8–32 mg/L to 0.25–1 mg/L; and for *C. parapsilosis*, it reduced from 0.125–32 mg/L to 0.03–0.125 mg/L. These dramatic reductions in MIC underscore the powerful potential of this combination to enhance the efficacy of itraconazole significantly.
In contrast to the clear synergies observed with azoles, the combinations of geldanamycin with both fluconazole and itraconazole against *C. auris* strains consistently resulted in an indifferent interaction, with FICI values ranging from 0.75 to 2. This suggests that geldanamycin does not potentiate the activity of azoles against this emerging multidrug-resistant pathogen under the tested conditions. Similarly, when geldanamycin was combined with either caspofungin or anidulafungin, representing the echinocandin class of antifungals, the interactions were consistently indifferent against all the *Candida* strains included in this study, with FICI values ranging from 0.562 to 3. Critically, and of significant positive implication for future therapeutic strategies, no instances of antagonism were detected across any of the studied combinations, indicating that combining geldanamycin with these antifungals does not lead to a reduction in their individual efficacy.
Discussion
The emergence of antifungal drug resistance, coupled with a notable increasing trend in infections caused by intrinsically resistant *Candida* species and the alarming global spread of the multidrug-resistant pathogen *C. auris*, poses an urgent and formidable threat to global public health, significantly complicating the effective treatment of *Candida* infections. Given the severely limited classes of antifungal drugs currently available in the clinical arsenal, there is a clear and pressing recommendation to vigorously pursue alternative and innovative therapeutic strategies. Among these, targeting key drivers of major intracellular mechanisms in fungi, such as the mammalian target of rapamycin (mTOR) proteins, calcineurin, and particularly Heat shock protein 90 (Hsp90), represents highly promising avenues to enhance antifungal activity and overcome resistance.
Hsp90 is a ubiquitous and essential molecular chaperone, comprising three distinct domains, each contributing to its diverse activities. Its N-terminal domain possesses an ATPase activity that is fundamental for the chaperone’s function, ensuring the proper folding, stability, and conformational maturation of a multitude of client proteins. The overexpression of Hsp90 is a common adaptive response observed in fungal cells when exposed to various environmental stresses, including elevated temperatures and oxidative stress. This upregulation serves as a crucial protective mechanism, safeguarding fungal cells and their essential proteins during unfavorable conditions, thereby contributing to their survival and pathogenicity. Considering the numerous and vital roles of Hsp90 in fungal biology, inhibitors of this protein have attracted considerable attention in various preclinical studies, animal models, and even clinical trials, particularly in the context of cancer therapy. Notably, Mycograb, a recombinant monoclonal antibody specifically targeting fungal Hsp90, has been shown to interact synergistically with amphotericin B against various *Candida* species. Furthermore, clinical and murine model studies have reported the superior efficacy of Mycograb combined with amphotericin B compared to amphotericin B alone, underscoring the translational potential of Hsp90 modulation.
Geldanamycin, a natural product first isolated from the fermentation broth of *Streptomyces hygroscopicus* in 1970, holds historical significance as one of the earliest identified Hsp90 inhibitors. Its mechanism of action involves competing with ATP for binding to the ATP-binding site located in the N-terminal domain of Hsp90, thereby disrupting the essential ATPase activity of this chaperone. While geldanamycin’s primary therapeutic interest initially centered on its potent antitumor activity, it has also demonstrated noteworthy antimicrobial properties. A prior study by Zhang et al. observed a synergistic interaction between geldanamycin and fluconazole against 8 out of 18 (44.4%) *C. albicans* isolates, while reporting indifferent interactions for *C. parapsilosis, C. glabrata, C. tropicalis*, and *C. krusei*. In our present study, despite finding indifferent interactions for combinations of geldanamycin with echinocandins, we observed striking and consistent synergies when geldanamycin was combined with azole drugs against *C. albicans, C. parapsilosis*, and *C. glabrata*. This synergistic effect was particularly pronounced with fluconazole plus geldanamycin, which notably demonstrated the ability to reverse fluconazole resistance in 100% of *C. albicans* isolates, and in 80% of *C. glabrata* and *C. parapsilosis* isolates. Furthermore, the combination of itraconazole with geldanamycin proved to be the most potent combination, exhibiting synergy against all (100%) *C. albicans, C. parapsilosis*, and *C. glabrata* isolates. The observed discrepancies in synergistic rates between our study and that of Zhang et al. can largely be attributed to variations in the susceptibility status of the isolates tested. In Zhang et al.’s work, nine out of eighteen *C. albicans* isolates were fluconazole-resistant, and synergy was seen in 66.6% of these. However, for the nine fluconazole-susceptible *C. albicans* isolates and all *C. parapsilosis* and *C. glabrata* isolates which were also susceptible, synergy was observed in only 22.2% of *C. albicans* isolates and not at all in the other two species. Our study utilized predominantly fluconazole-resistant isolates, which explains the significantly higher rate of synergistic interaction we observed, particularly the remarkable resistance reversal.
It is well-established that Hsp90 plays a crucial role in the emergence and maintenance of azole resistance in *Candida* by influencing the ergosterol biosynthesis pathway, which is the direct target of azoles. However, resistance mechanisms mediated by drug efflux pumps, which actively pump antifungals out of the cell, are generally not directly related to Hsp90 activity. A very recent study has shed light on *C. auris* by demonstrating that its high-level resistance to fluconazole is independent of Hsp90 and is primarily mediated by cdr-1 efflux transporters. This critical finding provides a clear explanation for the consistently indifferent outcome observed in our study for the combination of azoles with geldanamycin against fluconazole-resistant *C. auris* strains. The efflux mechanism effectively bypasses the Hsp90-mediated aspects of azole resistance that are present in other *Candida* species, thus rendering Hsp90 inhibition ineffective for *C. auris* resistance reversal.
Beyond drug resistance, Hsp90 is known to be intimately involved in various other pathogenic processes, including biofilm formation, morphogenesis, and the overall virulence of *C. albicans*, which remains the most common *Candida* species. Therefore, a synergistic combination that not only reverses drug resistance but also simultaneously impairs these fundamental pathogenesis factors would represent an exceptionally interesting and highly desirable hypothetical therapeutic outcome. However, the precise extent to which this multifaceted benefit is achieved needs further rigorous *in vitro* and *in vivo* investigation to be definitively clarified. It is also crucial to acknowledge that despite the promising preclinical data, the clinical translation of geldanamycin itself faces significant challenges. The binding site of geldanamycin on Hsp90 is highly conserved between human and fungal Hsp90, which means that geldanamycin exhibits considerable affinity for human Hsp90. This lack of selectivity leads to an immunosuppressive nature, as Hsp90 is critical for the function of many human immune proteins, posing a substantial hurdle for its systemic clinical use as an antifungal agent. Furthermore, as an anticancer compound, geldanamycin demonstrated poor water solubility and exhibited notable hepatotoxicity in animal models, leading to its non-evaluation in clinical trials for cancer. Nevertheless, geldanamycin has provided a foundational understanding that has spurred the development of new anticancer derivatives, such as 17-AAG (17-allyl-17-demethoxygeldanamycin) and 17-DMAG (17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin), which have successfully entered clinical trial phases, albeit for cancer. This success in anticancer drug development provides a valuable precedent. The same rational drug design approach can be applied to develop geldanamycin derivatives specifically tailored for fungal Hsp90, aiming to achieve improved antifungal activity with reduced host toxicity. In this regard, despite the high-level sequence homology between *Saccharomyces cerevisiae* Hsp90 and human Hsp90, specific conformational differences have been reported, providing structural insights that can be exploited for the rational design of fungal-specific inhibitors. Indeed, recent work by Whitesell et al. has already described an inhibitor of fungal Hsp90 that demonstrates a remarkable 25-fold selectivity for binding to fungal Hsp90, as determined in *C. albicans*. Accordingly, future studies focused on developing highly fungal-specific Hsp90 inhibitors or geldanamycin derivatives with significantly reduced immunosuppressive and toxic features hold immense promise to overcome the current clinical limitations and realize the full therapeutic potential of Hsp90 inhibition in managing invasive fungal infections.
Conclusion
In conclusion, our comprehensive *in vitro* investigation provides compelling evidence that the simultaneous targeting of Heat shock protein 90 (Hsp90) with geldanamycin and lanosterol 14-α demethylase, the molecular target of azole antifungals, represents a highly effective therapeutic approach against *Candida albicans*, *Candida glabrata*, and *Candida parapsilosis*. This dual targeting strategy demonstrated significant synergistic activity, leading to a profound reduction in the minimum inhibitory concentrations of azole drugs, which could potentially translate into enhanced clinical efficacy and the reversal of azole resistance in these species. However, it is crucial to highlight that this specific combined approach proved to be ineffective against the emerging and challenging pathogen *Candida auris*, underscoring its distinct resistance mechanisms that are not overcome by this particular combination. Furthermore, our study also determined that the combination of geldanamycin with echinocandin antifungals, specifically caspofungin or anidulafungin, resulted in consistently indifferent interactions across all studied *Candida* species, indicating a lack of synergistic or antagonistic effects. While these results offer valuable preclinical insights, it is important to acknowledge that they are based on a limited number of isolates. Therefore, further extensive studies, including those conducted *in vivo*, are critically required to confirm these findings across a broader range of clinical isolates and to rigorously evaluate the effectiveness and safety of these synergistic combinations in a more complex biological context, paving the way for potential clinical translation.