In this section, we will review the important HSP inhibitors which may be attractive therapeutic targets in cancer.
2.3.1. Targeting HSP27
HSP27 therapies are based on three distinct strategies—small molecule inhibitors, protein aptamers and antisense oligonucleotides (ASO) that bind to HSP27 and inhibits it function [
199,
200,
201]. Two small molecules as HSP27 inhibitors are currently under evaluation—quercetin and RP101 (
Figure 7A,B). Quercetin, a bioflavonoid compound, has been shown to have anti-tumor activities via targeting the HSF1-dependent HSPs in many cancer cell lines [
202,
203,
204,
205,
206,
207,
208,
209]. In NSCLC cell line A549, quercetin has been shown to inhibit HSP27 leading to reduced cell viability. Moreover, combination of quercetin with chemotherapeutic agents such as cisplatin or gemcitabine in the same cell line exhibited more potent cytotoxic activity [
210]. Although, quercetin has been proved to be a potent chemo-sensitizer and was involved in clinical trials for treatment of non-malignant diseases [
211], there are no on-going anti-cancer clinical trials involving this inhibitor. RP101, an antiviral nucleoside also known as bromovinyldeoxyuridine, BVDU, and brivudine, binds with HSP27 to inhibit its function [
201]. Similar to quercetin, this also acts as a chemosensitizing agent to prevent development of resistance [
212]. In pilot study involving stage III and IV pancreatic cancer patients, RP101 increased the survival by 8.5 months compared to historical controls and has recently concluded a phase II clinical trial in combination with chemotherapy agent gemcitabine (NCT00550004) [
201]. Although the trial was discontinued due to side effects caused by gemcitabine, no side-effects of RP101 was reported and development of second generation derivative of RP101 is currently underway [
212]. TDP, also known as 1,3,5-trihydroxy-13,13-dimethyl-2
H-pyran [7,6-b] xanthone and isolated from Chinese traditional medicinal herb
Garcinia oblongifolia, was also shown to downregulate of HSP27 expression leading to HSP27-mediated apoptosis and reduction of tumor cell growth in hepatocellular carcinoma [
213]. The second approach to target HSP27 utilizes peptide aptamer that binds to and disrupts the dimerization and oligomerization of HSP27 [
200]. Currently, two peptides, PA11 and PA50, are under investigation and very effective in sensitizing cancer cells to other therapies. In addition, these peptide aptamers significantly inhibited head and neck squamous cell carcinoma tumor growth in vivo via cell-cycle arrest [
200]. Although PA50 was more potent in vitro, it lacked the in vivo efficacy of PA11 that lead to significant cell death [
200]. The pre-clinical success of these peptides suggest that these peptides hold clinical promise. The third approach to target HSP27 involves the use of antisense oligonucleotide, OGX-427, which is shown to decrease HSP27 expression [
214]. In combination with chloroquine, OGX-427 has been reported to decrease prostate cancer xenograft (PC-3) tumor volume by two-fold after seven weeks of treatment compared to chloroquine alone treatment [
215]. It has also shown to inhibit HSP27 leading to sensitization of NSCLC cells to erlotinib and chemotherapy [
216] and was found more potent in combination with gemcitabine in case of pancreatic cancer [
217]. OGX-427 has also been tested in a phase I trial involving patients with metastatic bladder cancer (NCT00959868), a phase II trial (NCT01120470) involving castrate resistant prostate cancer patients in combination with prednisone, and in another phase II trial in combination with a successful CRPC drug—abiraterone (NCT01681433) and with promising outcomes. Seven additional clinical trials with OGX-427 are currently ongoing.
2.3.4. Targeting HSP70
Unlike normal cells, most malignant cells aberrantly express HSP70 to endure the multitude of insults at different stages of tumorigenesis as well as therapeutic treatment. This addiction for HSP70 in malignancy serves as the rational for HSP70 targeting in cancer therapy. A large amount of progress has been made in the last decade towards the development of HSP70 inhibitors. Here we review the recent advancement in development of HSP70 inhibitors and different strategies for their use in cancer therapeutic strategies. HSP70 family consists of several chaperone proteins with multiple cellular location (
Table 1) as well as distinct tissue expression. Structures of all members are similar that consists of two important domains therapeutically—the nucleotide-binding domain (NBD) and the substrate-binding domain (SBD). The ATPase pocket is present in the NBD and binds the J-domain containing proteins such as HSP40 to promote its ATPase activity (
Figure 8A) [
228]. The SBD contains the EEVD amino acid sequence motif that binds client protein to promote specific protein folding function to prime the cytoprotection in response to stress [
229]. HSP70 inhibitors can be divided into three basic categories—small molecular inhibitors, protein aptamers, and antibody treatment.
Although, development of small molecule inhibitor against HSP70 has yet to succeed to date, with only one entering clinical trial, we will describe the several inhibitors here in this section. First, 2-phenylethynesulfonamide (PES) or pifithrin-µ (
Figure 8A), a small molecule inhibitor binds to the C-terminal PBD of HSP70 disrupting its association with co-chaperone HSP40 as well as several clients including pro-apoptotic APAF-1 and p53 [
230]. This leads to aggregation of misfolded proteins, lysosomal membrane destabilization and apoptosis. PES is a potent antitumor agent in vitro and in vivo [
230]. PES has also shown potent cytotoxicity in various leukemia cell lines when administered in combination with SAHA (vorinostat) or an HSP90 inhibitor, 17-AAG [
231]. 15-DSG [15-deoxyspergualin) (
Figure 8B) is a natural immunosuppressive agent that can target the HSP70-NBD and APT interaction to disrupt HSP70’s ATPase activity [
232]. More potent activity was observed with second generation inhibitors such as MAL3-101 (
Figure 8C) and its derivatives, which was reported to disrupt the HSP70 ATPase activity blocking the proliferation properties of SK-BR-3 cancer cells [
233]. These inhibitors were observed to be minimally effective as monotherapy, but when used in combination with other agents was found to be very effective. MAL3-101 showed potent efficacy in combination with 17-AAG in melanoma cells and in combination with PS-341 (bortezomib) in a mouse model containing same cancer cell type [
234]. The cytotoxic activity of a proteasome inhibitor, MG-132 was also augmented when combined with MAL3-101 in primary multiple myeloma cells [
235]. VER-155008 (
Figure 8D), an adenosine derived compound, can also inhibit HSP70 function by attacking the ATPase domain. In vitro studies in BT474 breast cancer cells and HCT116 colon cancer cell line revealed that VER-155008 was able to induce caspase-dependent cell death and non-caspase-dependent cell death, respectively [
236]. VER-155008 also showed improved cytotoxic effects when combined with HSP90 inhibitors such asNVP-AUY922 in myeloma cells [
237] or 17-AAG in colon cancer cells [
236]. In addition, Azure C, myricetin and methylene blue are also identified as HSP70 inhibitors, but their specificity needs further validation [
238]. Finally, the only HSP70 small molecule inhibitor that has entered a clinical trial is MKT-077 (
Figure 8E), which is a cationic rhodacyanine dye analog and can also disrupt the ATPase domain of HSP70. Strong cytotoxic efficacy in vitro and in vivo promoted this agent to a phase I clinical trial, but due to nephrotoxic side effects the trial was halted claiming further investigation [
239].
In a second approach, aptamers were developed which bind to both SBD and NBD in order to attenuate HSP70 functions [
240]. A17, so far the most potent aptamer was shown to disrupt HSP70 function by attacking the NBD in in vitro biochemical analysis [
241]. In combination with cisplatin, A17 aptamer augmented cell death significantly in HeLa cells in vitro and led to tumor-free state in mouse model carrying B16F10 melanoma cells [
241].
The third and most promising strategy to develop HSP90 inhibitor is the advent of immune system based monoclonal antibody, cmHsp70.1, which recognizes and binds the extracellular motif—TKDNNLLGRFELSG (TDK) of membrane bound HSP70 [
242]. When colon cancer mouse model (CT26) was administered with cmHsp70.1 alone, it resulted in significant reduction in tumor weight and volume and survival rate was enhanced by 20% in 20 days [
242]. It has successfully completed the phase I trial [
243] and is currently evaluated in combination with chemoradiation therapy in phase II trial involving NSCLC patients (NCT02118415). Finally, there are other agents that being actively evaluated in different phases of development as described in
Table 2.
In addition, the immunogenic properties of HSP70 have made it a critical part of vaccine development. Several vaccines consisting of disease specific epitopes and HSP70 DNA have been made and clinically tested. A phase I clinical trial tested the feasibility and toxicity of vaccination made with HSP70 for the treatment of chronic myelogenous leukemia in the chronic phase (NCT00027144). Another vaccine is pNGVL4a-Sig/E7(detox)/HSP70 DNA which was clinically tested in patients with cervical intraepithelial neoplasia (NCT00121173) [
244]. Recombinant 70-kD heat-shock protein has been also used in a trial to treat chronic myelogenous leukemia in chronic phase (NCT00030303). More recently, a clinical study involving Natural Killer (NK) cell based adoptive Immunotherapy is recruiting participants for the treatment of NSCLC patients after radiochemotherapy (RCT), where they will employ Hsp70-peptide TKD/IL-2 activated, autologous NK cells (NCT02118415).
2.3.5. Targeting HSP90
The HSP90 family constitutes the most studied family of HSPs as many of HSP90 clients are involved in development and promotion of cancer. In this section, we will focus on the development of HSP90 inhibitors as cancer therapeutic agents. HSP90 targeting in cancer treatment started with natural inhibitor, geldanamycin (GM) (
Figure 9(AI)), which was derived from
Streptomyces hygroscopicus and exhibits potent antiproliferative activity via binding to the ATP-binding site of HSP90 to prevent its function [
251,
252]. Although it was able to induce potent in vitro and in vivo cytotoxic effects, due to its structural instability and hepatotoxicity, its phase I clinical trial was suspended and it failed to progress further [
253]. Despite the lack of success in the clinic, GM still plays fundamental roles as a potent HSP90 inhibitor for in vitro studies, especially in ERBB2+ breast cancer cells [
254,
255]. Another important natural inhibitor of HSP90 is radicicol (RD) (
Figure 9(AII)), which was derived from
Monosporium bonorden, which showed strong in vitro antitumor properties via attacking the core ATP-binding pocket of HSP90, but was proven ineffective in vivo due to its structural instability [
256]. In order to overcome these initial problems, GM analogues were developed. The first two important GM derivatives tested in the clinic are 17-AAG (also known as tanespimycin or 17-allylamino-17-demethoxygeldanamycin) and 17-DMAG (also known as alvespimycin or 17-dimethylaminoethylamino-17-demethoxygeldanamycin). The first HSP90 inhibitor to be evaluated in clinical trial was 17-AAG (
Figure 9(AIa)) in 1999 [
257]. Unfortunately, its development was limited by poor solubility and oral bioavailability. 17-DMAG (
Figure 9(AIb)), on the other hand, demonstrated potent anti-tumor activity and improved water solubility, which led to its involvement in various clinical phase I trials; however, dose limiting side effects were still present [
258,
259]. A next generation GM derivative developed was IPI-504 (also known as retaspimycin hydrochloride) (
Figure 9(AIc)), a reduced form of 17-AAG, which showed greater promise as it had improved water solubility. It was evaluated in multiple phase I/II trials, some of which are still ongoing [
27,
251,
260,
261]. A nonquinone GM derivative that showed strong HSP90 binding property and much less side effects was WK88-1 (
Figure 9(AId)) [
262]. Finally, the most advanced and potent HSP90 inhibitors were developed as second generation derivatives of RD. First in this class is NVP-AUY922 (also known as luminespib or VER-2296] (
Figure 9(BI)), which showed strong efficacy both pre-clinically and clinically [
263,
264,
265]. Another second generation, radicicol derived agent is AT13387 (also known as Onalespib) (
Figure 9(BII)). One of the most important characteristic of this inhibitor is its prolonged pharmacodynamics action as it suppressed the EGFR signaling for a considerable period of time in vivo in NSCLC [
266]. Promising pre-clinical data led to the involvement of this drug in multiple phase I/II clinical trials, some of them are completed (NCT01294202, NCT01685268, NCT00878423, NCT01246102), and other are recruiting.
By far, the most promising synthetic, resorcinol-based, second generation HSP90 inhibitor was ganetespib (STA-9090) (
Figure 9(BIII)), which binds to the N-terminal APT-binding pocket of HSP90 disrupting the chaperone cycle. It is a small molecule inhibitor that contains a triazole moiety [
8,
267]. The potent anti-tumor activity of this HSP90 inhibitor has been translated from preclinical success into several clinical studies. Ganetespib has demonstrated its efficacy not only in monotherapy, but also in combination with other drugs in various cancer types driven by different oncogenic mutations such as mutant
EGFR [
268] and
KRAS mutant NSCLC [
269,
270,
271]. Ganetespib produced significant single agent activity in ALK-driven disease, however only transient responses were reported in patients with
KRAS mutant tumors due to development of rapid acquired resistance [
272]. Moreover, a large scale phase III clinical trial (Galaxy-2) in advanced lung cancer examining the combination of ganetespib and docetaxel failed to demonstrate either a PFS or OS benefit in either
KRAS mutant or
KRAS wild type NSCLC patients [
273]. Recent preclinical research in our laboratory has led to the discovery of the ganetespib resistance mechanism in
KRAS mutant NSCLCs. We have not only demonstrated that the acquired resistance to ganetespib in
KRAS mutant NSCLC is due to the hyperactivation of critical ERK1/2-p90RSK-mTOR signaling arc and subsequent bypass of G
2-M checkpoint arrest [
274,
275]. Moreover, we observed that ganetespib resistance led to cross resistance to the anti-microtubule agent, docetaxel, which could explain the failure of the Galaxy-2 trial [
274]. Although the failure of the Galaxy-2 trial led to cessation of any preclinical or clinical development of ganetespib, our preclinical analyses strongly suggest that the combination of ganetespib with an ERK1/2 inhibitor, or a p90RSK inhibitor or a CDC25C inhibitor would be an efficacious therapeutic strategy to test in the clinic [
274]. Based on our results, we suggested that one of these inhibitors tested in this study with ganetespib or another HSP90 inhibitor that is currently under clinical evaluation (e.g., AT13387, NCT01712217 and TAS-116, NCT02965885) may well prevent acquired resistance to HSP90 inhibitor and/or help overcome the resistance after HSP90 inhibitor monotherapy [
274].
X-ray crystallography studies advanced the rational designing of new second generation HSP90 inhibitors. Taking advantage of the new technologies, several purine and purine like analogues were generated that can effectively inhibit HSP90. CNF-2024/BIIB021 (
Figure 9(CI)) is a unique among the other members of this class, which has been evaluated in phase I/II trials. In a phase I trial involving patients with chronic lymphocytic leukemia, this drug caused dizziness in patients other grade 3 or 4 toxicities including fatigue, hyponatremia and hypoglycemia despite showing potent efficacy [
276]. It was also involved in another phase I study involving HER2+ metastatic breast cancer and was planned for a phase II study, but further development of the product was halted due to strategic reason [
277]. Other important members of this family are Debio 0932 and PU-H71. Debio 0932 (
Figure 9(CII)) entered a phase I study in 2010 being evaluated in patients with advanced solid tumor or lymphoma and completed the first part (phase Ia, NCT01168752). A phase II study involving patients with NSCLC started but had to be terminated due to occurrence of dose limiting toxicities (NCT01714037). PU-H71 (
Figure 9(CIII)) has been evaluated in phase I clinical trial involving patients with lymphoma, advanced solid tumors, and myeloproliferative disorders and also being actively evaluated at the National Cancer Institute in patients with advanced solid tumors and low-grade non-Hodgkin’s lymphoma [
277].
SNX-5422 (
Figure 9D) is another important HSP90 that was discovered using an ATP-affinity column which contains a pyrazole ring [
278]. Although it was able to enter a phase I trial in 2007, the development was stopped due to ocular toxicity and the potential for irreversible retinal damage [
277]. The newest candidate to the stage is TAS-116 (
Figure 9E), that was discovered utilizing a multiparameter lead optimization campaign [
279]. It has been demonstrated to show potent cytotoxicity both in vivo and in vitro. It has been characterized to have favorable bioavailability and better metabolic stability in rodents and non-rodents and has also showed less ocular toxicity and exerted stronger anti-tumor activity in several xenograft models [
279,
280]. It has also been demonstrated to enhance radiosensitivity of human cancer cells to X-rays and carbon ion radiation in preclinical studies [
281], with a favorable clinical therapeutic index. A Phase IA/IB study evaluating TAS-116 has just been initiated (NCT02965885) involving patients with HER2
+ MBC, NSCLC harboring
EGFR mutations will be further evaluated for safety, tolerability and efficacy in 3 separate cohorts at recommended dose. Based on
clinicaltrials.gov, we have tabulated the different inhibitors that are being evaluated clinically either in mono or in combination therapy so far in
Table 3.