A novel sulfamethoxazole derivative as an inhibitory agent against HSP70: A combination of computational with in vitro studies

Abstract

In the current study, a novel derivative of sulfamethoxazole (a sulfonamide containing anti-biotic) named ZM-093 (IUPAC name: (E)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)diazenyl)-N-(5-methylisoxazole-3-yl)benzenesulfonamide) was synthesized via common diazotization-coupling reactions from sulfamethoxazole and subsequently characterized through NMR/FT-IR spectroscopy. After evaluation, the compound was geometrically optimized at the DFT level of theory with BL3YP method and 6/31++G (d,p) basis set and from the optimized structure, several molecular descriptors important in the biological reactivity of the compound, such as global reactivity parameters, molecular electrostatic potential, average local ionization energy, and drug-likeness features of the compound were computationally analyzed. The experimental in vitro investigations of the interaction between ZM-093 and heat shock protein 70 (HSP70), a protein that is highly expressed in several types of cancers, exhibited a significant inhibitory effect against the chaperone activity of HSP70 for the titled compound (P-value < 0.01) and the comparison between the experimental studies with the mentioned computational analysis, as well as molecular docking, illustrated that ZM-093 may inhibit HSP70 through binding to its substrate-binding domain. Finally, by taking all the previous results into account, a new method for assessing the inhibitory activity of ligand to HSP70 is introduced based on protonography, a recently developed method that is dependent on the catalytic activity of carbonic anhydrase on polyacrylamide gel electrophoresis.

Introduction

Sulfonamides or sulfa-drugs are key motifs in pharmaceuticals that have drastically influenced the pharmacological markets in the recent century [1]. They are composed of a sulfur atom that has two sets of double bonds to two oxygen atoms, a carbon-based side group, and a nitrogen atom bonded to the sulfur itself [2]. The formation of hydrogen bonds in sulfonamides increases both their solubility and their ability to interact with their targets [3]. Several types of sulfonamides include phenolic sulfonamides [4], hetaryl sulfonamides [5], benzenesulfonamides [6], sulfonamide-containing pyrazoles [7], et cetera and there is a plethora of applications for sulfa-drugs in medicinal chemistry. Sulfonamide-based compounds are heterocyclic molecules that have exhibited several biological activities including their role as antibacterial [8], antifungal [9], anti-inflammatory [10], carbonic anhydrase inhibitors [11], lactoperoxidase inhibitors [12], acetylcholinesterase inhibitors [13], and anticancer agents [14]. Sulfonamide derivatives can act as anticancer agents through multiple mechanisms such as inhibition of tubulin polymerization [15], PI3K [16], Cox-2 [17], Akt [18], histone deacetylase [19], and NADPH reductase [20]. Sulfamethoxazole is an aminobenzene sulfonamide bacteriostatic antibiotic that is frequently used in combination with trimethoprim as the drug Bactrim [21]. Sulfamethoxazole competitively inhibits dihydropteroate synthase preventing the formation of dihydropteroic acid, a precursor of folic acid which is required for bacterial growth [22].

Mammalian heat shock proteins (HSPs) are a family of proteins, traditionally known as molecular chaperones, that can reverse or inhibit the denaturation of cellular proteins in response to cellular stress such as high temperature [23]. HSPs are sorted into several classes according to their monomeric molecular mass: First group includes HSP110, HSP100, HSP90, HSP70, and HSP60 which are ATP-dependent and are referred to as high molecular weight HSPs. The other group which is called low molecular weight HSPs has a molecular weight of 34 kDa or less and act in an ATP-independent manner [24], [25]. HSP70 is a member of the molecular chaperone family that is found in every membranous organelle of nearly all cells. The expression of HSP70 is usually kept at low levels under physiological conditions but is induced in protein-damaging situations such as heat shock, oxidative stress, hypoxia or heavy metals in order to provide resistance to proteotoxic stresses [26]. The main roles of HSP70 include protein folding as well as disaggregation and degradation of misfolded proteins. The HSP70 proteins contain two major functional domains: N-terminal nucleotide-binding domain (NBD) (45-kDa) and C-terminal substrate-binding domain (SBD) (25-kDa) [27], [28]. The binding of ATP to its corresponding domain facilitates the binding and releasing of protein substrate to HSP70 [29]. The presence of HSP40 proteins (also referred to as DnaJ or J-domain proteins) is necessary for the function of HSP70 since they stimulate the ATPase activity of HSP70 through their J-domain and also bind to non-native proteins with the purpose of presenting them to HSP70 [30]. The function of HSP70 has been linked to neurodegenerative diseases (e.g. Alzheimer's disease) and various types of cancers [31], [32]. Overexpression of HSP70 leads to an increase in resistance against apoptosis-inducing drugs such as imatinib, and doxorubicin and therefore, is associated with cancer [33], [34]. Moreover, HSP70 has also been shown to be able to reduce radiation sensitivity in tumor cells [35] and its phosphorylation may increase the resistance of tumor cells against methotrexate [36]. On the other hand, down-regulation of HSP70 increases the vulnerability of tumor cells to apoptosis [37]. Thus, HSP70 is an anti-apoptotic protein that can block both the extrinsic (death receptor) and intrinsic (mitochondria-dependent) pathways of apoptosis [38].

Highly-expressed HSP70 can interfere at three levels in apoptotic signaling: upstream to mitochondria, at the mitochondria, and post-mitochondria [39]. Release of cytochrome c from intermembrane space of mitochondria via apoptotic signals induces the ATP-dependent formation of Apaf-1-caspase-9 apoptosome which in turn activates caspase-9 [40], [41]. HSP70 can block the release of cytochrome c by preventing the translocation of Bid and Bax to the mitochondria [42]. HSP70 is also able to directly bind to Apaf-1, thereby hindering the activation of procaspase-9 [43]. Moreover, it has been indicated that the interaction of HSP70 with procaspase-7 and -9 suppresses their proteolysis [44]. Apart from caspase-dependent apoptosis, HSP70 can also associate with apoptosis-inducing factors and block AIF nuclear translocation which results in AIF-induced chromatin condensation [45].

Several reasons have made HSP70 become a well-liked target for drug discovery for various types of cancer. These include the predescribed importance of HSP70 dysregulation in cancer, the abundance of HSP70 family in almost every human tissues [26], the existence of various isoforms of HSP70 in human's genome [46], and similarity of structural domains in HSP70 isoforms [47] and according to these basis, several strategies have been proposed in order to inhibit the function of HSP70 [48], [49]. The first attempt was approached by Hosokawa et al. via the inhibition of the transcriptional factor of HSP70, HSF1 [50]. Another procedure described by Guo et al. was to knockdown HSP70 gene using siRNAs [51] but HSP70 itself has two potential inhibitor-binding sites based on its structure: the ATP binding site and the substrate-binding cleft [52]. 2-Phenylethynesulfonamide (also known as pifithrin-μ) and AEAC (a synthetic derivative of colchicine) are among small molecules that bind to the substrate-binding domain [53], [54] whereas apoptozole and artesunate can bind to the ATPase domain [48], [55].

Studying the inhibitory effects of apoptosis-inducing compounds on HSP70 activity is not plain and demands laboratory approaches such as X-ray crystallography, fluorescence polarization, and surface plasmon resonance [56]. Despite the high sensitivity of these techniques, they are costly and time-consuming due to long sample preparation steps before analysis. Hence, providing an accelerated, sensitive, and inexpensive method for screening apoptosis inducers based on inhibition of HSP70 is pivotal. Cell lysis and total protein extraction are considered the main parts for most of the techniques in order to study the chaperone activity of proteins [57]. To tackle this issue, in 1993 Schröder, et al. employed an approach for real-time analysis of intracellular proteins based on a co-expression system made up of a chaperone and a reporter gene in Escherichia coli [58]. Later, a hypothesis for the role of HSPs in protecting reporter proteins was put forward by Forreiter, et al. in 1997 [59]. In a real-time reporter system, luciferase is a promising candidate as an intracellular reporter in a bioluminescence assay due to the reproducibility, rapidity, and sensitivity of the assay and the lack of a physiological role for luciferase in E. coli metabolism [60]. Recently, for the first time, a real-time reporter system has been introduced for screening apoptosis-inducing compounds in E. coli cells based on inhibition of the activity of Hsp70 [57].

Carbonic anhydrases (CAs), are a class of proteins capable of catalyzing the hydration of carbon dioxide to bicarbonate and protons: CO₂ + H₂O ↔ HCO₃^- + H⁺ [61]. It has been shown that HSP70 can proceed with the folding process of CAs through their chaperone activity [55]. Protonography is a new derivative method of zymography, based on the activity of CA on SDS-PAGE gel. The produced protons through the function of CA are responsible for the change of the color which appears on the gel in the corresponding band. The boldest advantage of this technique is that based on the molecular weight markers, the activity of CAs with different molecular weights can be detected and quantified rapidly on a single gel [62], [63]. So far, protonography has been employed for a variety of uses related to different classes and isoforms of CA. These include analyzing the enzymatic activity of mammalian CA, α-CA, η-CA, and ɩ-CA on the protonogram [62], [64], [65], studying the oligomeric state of γ-CA (from Porphyromonas gingivalis) [66], investigating the inhibitory activity of inorganic anions against α-CA (from Corallium rubrum) [63], and studying the efficiency of sulfonamide derivatives against α-CA, β-CA, γ-CA, and ɩ-CA [67], [68], [69]. Since HSP70 can facilitate the folding process of CA, inhibition of HSP70 suppresses the progress of CA folding which can consequently be measured by protonography. In the present study, we have examined the effect of a novel sulfonamide derivative on HSP70 activity through a new protonography based method. In this study a sulfamethoxazole derivative called ZM-093 with IUPAC name of (E)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)diazenyl)-N-(5-methylisoxazole-3-yl)benzenesulfonamide was synthesized, evaluated, computationally analyzed and its effect on the chaperone activity of HSP70 was assessed.