Proteasome inhibitor patents (2010 - present).

Review

Proteasome inhibitor patents (2010 -- present) Rainer Metcalf, Latanya M Scott, Kenyon G Daniel & Q Ping Dou† †

Wayne State University, Karmanos Cancer Institute, Detroit, MI, USA

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Overview

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Current proteasome inhibitors: novel chemical entities created specifically for inhibiting the

Expert Opin. Ther. Patents Downloaded from informahealthcare.com by University of Washington on 08/28/14 For personal use only.

proteasome 3.

Combination therapy: known or novel compounds used in combination with other known drugs for proteasome inhibition and synergy of therapeutic effect

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Expert opinion

Introduction: Over the past 3 years, numerous patents and patent applications have been submitted and published involving compounds designed to inhibit the proteasome. Proteasome inhibition has been of great interest in cancer research since disruption of proteolysis leads to a significant buildup of cytotoxic proteins and activation of apoptotic pathways, particularly in rapidly proliferating cells. The current standards in proteasome inhibition are the only FDA-approved inhibitors, bortezomib and carfilzomib. Although these drugs are quite effective in treating multiple myeloma and other blood tumors, there are shortcomings, including toxicities and resistance. Most of the current patents attempt to improve on existing compounds, by increasing bioavailability and selectivity, while attempting to reduce toxicity. A general categorization of similar compounds was employed to evaluate and compare drug design strategies. Areas covered: This review focuses on novel compounds and subsequent analogs developed for proteasome inhibition, used in preventing and treating human cancers. A comprehensive description and categorization of patents related to each type of compound and its derivatives, as well as their uses and efficacies as anticancer agents is included. A review of combination therapy patents has also been included. Expert opinion: Although there are many diverse chemical scaffolds being published, there are few patented proteasome inhibitors whose method of inhibition is genuinely novel. Most patents utilize a destructive chemical warhead to attack the catalytic threonine residue of the proteasome active sites. Few patents try to depart from this, emphasizing the need for developing new mechanisms of action and specific targeting. Keywords: anticancer therapy, bortezomib, carfilzomib, clinical trials, drug discovery, patents, polyphenols, proteasome inhibitors Expert Opin. Ther. Patents (2014) 24(4):369-382

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Overview

Protein turnover is an essential part of amino acid metabolism in which cells constantly synthesize and degrade proteins. The rate of expression and degradation of proteins is critical for the proper regulation of metabolic pathways. The proteasome is a nearly universal cellular component and integral for this fundamental biological process [1,2]. The 26S mammalian proteasome is a massive 2400 kDa molecule comprising a 20S core particle (CP) and one or two 19S 18-subunit regulatory particle (RP) [3]. The RP, or PA700 cap, also incorporates specific recognition sites to which ubiquitin chains can bind [4]. The CP is a 700 kDa, barrel-shaped structure composed of four six-subunit rings [5]. The a subunits of the outer rings are largely structural in function and appear to regulate the formation and stabilization of either the 26S proteasome or 19S immunoproteasome [6]. Proteolysis occurs at the six protease active sites of the inner ring b subunits oriented toward the lumen

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Article highlights. . . .


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Proteasome inhibition remains an effective technique in cancer therapy. A few recent proteasome inhibitor patents are genuinely novel in their method of inhibition. Most patents attempt to utilize epigallocatechin gallate or a peptidomimetic moiety coupled with a chemically reactive warhead. None of the currently published compounds appear to have the optimal combination of potency, toxicity and bioavailability.

This box summarizes key points contained in the article.

of the proteasome [3]. These active sites comprise a catalytic region and multiple recognition regions [7]. The catalytic regions are similar and incorporate a catalytic threonine 1 (Thr 1) residue to execute the hydrolysis of the protein [3]. Discretionary binding of specific residue sequences is accomplished by the recognition regions of the active sites [8]. These sites are designated as the b1/PRE3peptidylglutamyl peptide caspase-like recognition site (PGPH), the b2/PUP1 trypsinlike recognition site (T-L) and the b5/PRE2 chymotrypsinlike recognition site (CT-L) [9]. Other structural variants of the proteasome have been discovered and they exhibit slightly altered functions of the b subunits and regulatory caps. The immunoproteasome consists of smaller 11S RPs and a modified 20S CP containing ib1, ib2 and ib5 subunits and structural isoforms of the constitutive b1, b2 and b5 subunits, respectively [10]. The thymoproteasome is another proteasomal polymorphism more recently elucidated, which incorporates the ib1 and ib2 immunoproteasome subunits but integrates a constitutively differing tb5 subunit [11]. Disruption of proteolysis can lead to significant buildup of cytotoxic proteins and activation of apoptotic pathways, particularly in rapidly proliferating cells [12]. For this reason, proteasome inhibition has been of great interest in cancer research [13]. Further studies into the use of proteasome inhibitors on oncogenic cell lines have revealed that they may prevent angiogenesis and metastasis and increase susceptibility to apoptosis, while quiescent cells may be preserved to some extent [14]. Possible therapies for other diseases and disorders, such as neurodegenerative disorders [15], cardiac disease [16,17] and organ transplant rejection [18], are being researched. The patent search strategy was carried out with focus on patents or applications from the past 3 years that cover new chemical entities, or new methods of using natural products, known chemical entities, or any combinations of the aforementioned as inhibitors of proteasome activity. Additionally, the scope of utility of these patents was not limited to only cover cancer therapy but included patents that teach proteasome inhibition as a method of treating other diseases, such as immunological disorders or viral infections. The search 370

was initiated broadly to capture the most patents and applications filed between 1 January 2010 and 30 September 2013, using the Delphion patent database from Thomson Reuters. This search focused on finding representative classes of new chemical entities being claimed as novel proteasome inhibitors.

Current proteasome inhibitors: novel chemical entities created specifically for inhibiting the proteasome

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Proteasome inhibitors can be categorized into several different chemical classifications. A recent review article written by our group outlined a generalized list of current proteasome inhibitors based on chemical structure and reaction mechanism [19]. Many patents tend to circulate around existing structures, seeking to improve potency and selectivity. These analogs possess a similar pharmacophore comprising two key elements: a peptide portion that selectively binds to the recognition binding pocket of the proteasome with high affinity and a chemical moiety specifically designed to interact with the catalytic Thr 1 residue to irreversibly inhibit protease activity [20]. Common functional groups that are exploited to react with the catalytic Thr 1 residue are aldehydes, vinyl sulfones, boronates, epoxyketones and b-lactones. Boron-containing compounds Boron-containing inhibitors capitalize on highly reactive boron-containing ‘warheads’ to create a covalent ligature to a protein. Therefore, these types of molecules are generally noncompetitive and extremely potent, with IC50 values often in the nanomolar and picomolar range. One of the most well-known proteasome inhibitors, bortezomib (Figure 1-1), is of this type, utilizing a boronic acid moiety to perform a nucleophilic attack on the CT-L catalytic Thr 1 residue. This mechanism is a frequently adopted design strategy with attempts geared mainly to decrease side effects. Although bortezomib is a potent and effective proteasome inhibitor, it has been found to have several and often severe side effects, including fatigue, nausea, sensory neuropathy and adverse cardiovascular effects [21,22]. Additionally, the development of bortezomib resistance in patients and preliminary studies showing limited effect on solid tumors are being investigated [23-26]. Li et al. developed multiple compounds directed primarily at alleviating the side effects of bortezomib. The most active compound, 6f (Figure 1-2), displayed a CT-L IC50 of 0.079 ± 0.011 µM in biochemical assays, as well as an IC50 of 3.630 ± 1.669 µM in the PGPH-active site [27]. It also exhibited greater efficacy in cellular assays than bortezomib at 0.5 µM across multiple cell lines: HL-60 human leukemia with 87% inhibition, BGC-823 human gastric cancer with 95% inhibition, bel-7402 human hepatocarcinoma with 94% inhibition and Kb human nasopharyngeal carcinoma 2.1

Expert Opin. Ther. Patents (2014) 24(4)


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Figure 1. Boron-containing compounds are shown.

with 96% inhibition. Further, in vivo testing showed a 55% tumor inhibition [27]. Unfortunately, a 63% survival rate resonated as on par with the toxicity of bortezomib [27]. Compound 6g (Figure 1-3), in contrast, presented an efficacy comparable to bortezomib in biochemical and cellular assays, but with a 100% survival rate in rat models [27].

An interesting approach to the use of a chemical warhead by utilizing a type of protecting group on the warhead can be illustrated in the patent by Bernardini et al. [28]. This protecting group serves two purposes: one being that it masks the polarity of the highly polar boronic acid moiety, thereby increasing bioavailability, and two, it attempts to decrease


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Figure 2. Epoxyketone-containing compounds are shown.

the reactivity of the warhead until binding, conceivably reducing off-target effects. The patent outlined a large array of example compounds, but the general structure shown in Figure 1-4 characterizes the basic form of the protecting group on the boronic acid warhead. The group did a simple high-throughput screening to ascertain IC50 and EC50 values for each compound [28]. Compounds with the general form in Figure 1-4 displayed biochemical assay IC50 values of < 10 nM and EC50 values of < 200 nM in Molt 4 cells [28]. The compound shown in Figure 1-5 is a specific example, representative of the general form (Figure 1-4). Other recent patents on bortezomib analogs include compounds I-20 (Figure 1-6) [29], with a reported IC50 372

of < 50 nM and a list of compounds with the general structure shown in Figure 1-7 by Shenk et al. [30]. Another patent of note is on distinct formulations for stabilizing bortezomib in a liquid form suitable for injection [31]. While this patent may not seek claim for a specific compound, it does show alternative patent strategies in the realm of drug design. Epoxyketones Epoxyketones garnered significant interest after the introduction of carfilzomib (Figure 2-1) as a safer, more specific and more effective proteasome inhibitor than bortezomib [32]. Carfilzomib was designed as an analog to epoxomicin based on its in vivo antitumor activity [33]. The epoxyketone 2.2




pharmacophore is the main chemical moiety developed to attack the CT-L catalytic Thr 1 residue by forming a dual covalent morpholino adduct [34]. Compound 1 (Figure 2-2) was patented by Kirk and Jiang and showed that dosages of 30 -- 40 mg/kg reduce the number of visible metastatic tumors in lungs by ~ 50% [35]. Shenk et al. applied for a patent on compound 14 (Figure 23) specifically as a potential treatment for rheumatoid arthritis. Their work showed that dosages at 6 mg/kg reduced arthritis symptom severity in rat models by 50% [30,36]. Smyth et al. published multiple patents focusing solely on the structure and synthesis of compounds that emphasize a diversity of peptide mimetics employing an epoxyketone warhead [37-39]. The general structure is shown in Figure 2-4. Most proteasome inhibitors target the hydrophobic CT-L site since the majority of pharmaceutically viable compounds must be relatively hydrophobic themselves. This leaves the other sites severely underresearched as targets. Kisselev et al. developed compounds with the intent of specifically inhibiting the T-L active site [40]. The compound NC-022 (Figure 2-5) displayed preferential binding to the T-L site with an IC50 of 38 nM [40]. Other analogs portrayed similar potencies; however, NC-022 was the most cell-permeable compound and, thus, held the greater potential as an applicable therapeutic [40]. The authors also found that vinyl sulfones tended to be more potent and more selective for T-L than epoxyketones with same sequence and showed that T-L inhibition sensitizes cells to CT-L inhibitors, leading to possible combination therapies mentioned in later sections [40]. Despite the success of carfilzomib, epoxyketones capitalize on the same basic mechanism as bortezomib and share many of the common patient inconveniences and side effects. Although Carfilzomib demonstrated a 22.9% overall response rate in clinical studies and showed reduced incidence of peripheral neuropathies compared to bortezomib [9,41], carfilzomib still presented some side effects including pneumonia, acute renal failure, pyrexia, congestive heart failure, cytopenia, fatigue, nausea and dyspnea [32,41]. Continued research and improvements on this family of compounds are necessary to yield more safe and effective therapeutic options. Green tea-derived polyphenolic compounds Epigallocatechin gallate (EGCG) (Figure 3-1) has gained considerable interest in cancer treatment due to evidence of its ability to inhibit growth of several types of cancers [42-49]. Other studies have also shown EGCG to have certain selective binding to the proteasome, making it a potential model for designing novel proteasome inhibitors [50]. EGCG belongs to a class of natural products known as flavonoids, commonly found in green tea and other edible plants [51]. The EGCG analogs, compounds 5 and 7 (Figure 3-2 and 3-3, respectively), studied by Chan et al. show promise in the treatment of multiple myeloma (MM) ARP cells with in vitro tests displaying 70% growth inhibition at 50 µM for 2.3

compound 5 and 95% growth inhibition for compound 7 at the same concentration [52]. Another patent by the same author endeavored to increase the bioavailability of EGCG by increasing the hydrophobicity of the compounds and decreasing the rate of metabolism by introducing protecting groups [53]. By acetylating all of the hydroxyl groups on EGCG, the authors were able to create a prodrug (compound 1, Figure 3-4) that exhibited slower metabolic degradation and greater cell permeability, while retaining the potency of EGCG [53]. Patents for synthetic polyphenolic compounds developed by our group show interesting improvements in efficacy by changing the stereochemistry of natural products. By switching the chirality of the natural compounds, (--)-EGCG (Figure 3-1) and (--)-GCG (Figure 3-5), to the opposite plane polarized forms, (+)-EGCG (Figure 3-6) and (+)-GCG (Figure 3-7), the authors were able to substantially increase potency from IC50 values of 205 nM for (--)-EGCG to 170 nM for (+)-EGCG, and 610 nM for (--)-GCG to 270 nM for (+)-GCG [54]. Unmodified flavonoids are generally weak inhibitors, with common IC50 values in the range of many micromolars. However this property, coupled with their low toxicity and antioxidant activities, allows flavonoid analogs to fulfill a unique niche as low-risk agents that could be incorporated into diet as part of a preventative strategy for disease. Metal complexes Metal complexes using gold [55,56], nickel [57,58], zinc [57,58] and to a greater degree copper [59-63], have received significant attention due to their unique pharmacophore. Copper complexes have been viewed with particular promise as potential proteasome inhibitors, in addition to a nascent strategy in cancer therapy, after studies showed that cancer cells accumulate more copper than healthy cells [59-63]. 8--hydroxyquinoline (8OHQ) (Figure 4-1) has been well known as a monoprotic bidentate chelating agent for some time [64], although its potential as a proteasome inhibitor was only recently elucidated when our group found that the 8OHQ analog, clioquinol (Figure 4-2), inhibited the CT-L activity of the proteasome, blocked proliferation and induced apoptosis in breast cancer cells, while remaining nontoxic to noncancerous breast cells [60]. Since then, there have been several novel inhibitor designs incorporating the basic 8OHQ structure. Recent analogs, 5-amino-8-hydroxyquinoline (5AHQ) (Figure 4-3), of 8OHQ by Schimmer include an additional amino group and has similar IC50 values as 8OHQ [65]. In vivo assays of 5AHQ against human leukemia displayed modest IC50 values of 3.7 ± 0.3 µM. Sheshbaradaran exploited the preformed gallium metal complex, tris(8quinolinolato)gallium(III) (Figure 4-4), at 2.5 -- 5 µM in combination with 5 nM concentration of bortezomib to show a near 100% growth inhibition of lung cancer tissue [66]. 2.4


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Figure 3. Green tea-derived polyphenolic compounds are shown.

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indicate a strong potential for a viable treatment of various cancers [73-76]. Natural products isolated from marine organisms have served as an exceptional source for unique lead compounds in drug design [77,78]; therefore, it is not surprising to see a representative of this class of compounds showing potential as a prime archetype for a new class of novel proteasome inhibitors. Salinosporamide will likely continue to be of interest to researchers and a robust pharmacophore in future drug design strategies.

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Unique chemical designs Whereas many patents utilize previously discovered structures as a pharmacophore for developing novel compounds, there have been some recent publications employing entirely original structures. The apparent majority of effective proteasome inhibitors are noncompetitive and make use of some sort of ‘warhead’ to covalently modify the active site threonine residues. Competitive proteasome inhibitors rarely achieve activities lower than several micromolars. However, Lawrence et al. discovered an interesting compound, PI-1833 (Figure 6-1), reporting an IC50 of 630 ± 350 nM. The group also tested multiple analogs with some (PI-1840, Figure 6-2) achieving reported values of 32 ± 0.15 nM, alluding to the possibility of having found the first competitive proteasome inhibitors comparable to bortezomib [79]. The same group also discovered another unique compound, HLM-008182 (Figure 6-3), with a reported IC50 of 650 ± 40 nM [80]. Another innovative patent by Bachovchin et al. [81] capitalizes on the overexpression of fibroblast activation protein (FAP) in stromal fibroblasts of epithelial cancers [82]. FAP binds to a specific peptide sequence attached to a cytotoxic moiety; Figure 6-4 is an example of one such peptide sequence. The enzyme cleaves the compound to release the active moiety only when present in cells with sufficient expression of FAP [81]. While the active moiety may not necessarily be a proteasome inhibitor, the patent includes many of the aforementioned warheads utilized as the active group, R, in Figure 6-5. The patent focuses primarily on the mechanism of FAP-activated compounds, allotting to the possible incorporation of any cytotoxic moiety for the treatment of epithelial carcinomas. Although the novelty of this patent is not technically in proteasome inhibition, its inclusion in this review is significant due to the patent’s unique attempt to specifically deliver a proteasome inhibitor to certain cancer cells. Even though these compounds have only recently been discovered and are a long way from the clinical use, further investigation and design improvement will no doubt yield compounds with considerable potential as therapeutic agents. In order to surmount the obstacles faced by these promising leads, an ongoing research effort is essential to understand how these compounds achieve such high potencies and possibly elucidate new pharmacophores for effective competitive proteasome inhibition. 2.6

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Figure 4. Metal complexes are shown.

Salinosporamide analogs The marine natural product, salinosporamide A (Figure 5-1), has recently received considerable notice mainly due to its remarkable potency as a proteasome inhibitor with IC50 values often reaching the low picomolar range. The g-lactam--blactone bicyclic core of salinosporamide A is the fundamental structure and also functions as a proteasome inhibitor by covalently modifying active site threonine residues [67-69]. The replacement of the chlorine group on salinosporamide A to a hydroxyl group (Figure 5-2) led to a substantial decrease in cytotoxicity, 38 ± 4 µM compared to 9.8 ± 3 nM, while retaining potency, CT-L IC50 of 14 ± 1.5 nM compared to 2.5 ± 1.2 nM, and increasing selectivity for the CT-L active site [70]. Various compounds with the general structure in Figure 5-3 were patented by Ling et al. [71]. The patent included a method for synthesizing the natural product and the structures of any intermediates formed in the process. The authors also compared the differences in inhibition between production through fermentation and synthetic manufacturing [71]. The synthetic form, being more pure, exhibited a slightly better CT-L IC50 of 3.2 nM and 96% inhibition of the 20S proteasome in RPMI 8826 cells at 10 nM concentrations [71]. Although the natural form of salinosporamide A cannot be patented, Fenical et al. have applied for a patent on multiple stereoisoforms of salinosporamide [72] and early clinical trials of the natural compound, clinically named marizomib, 2.5


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Figure 5. Salinosporamide analogs are shown.

3. Combination therapy: known or novel compounds used in combination with other known drugs for proteasome inhibition and synergy of therapeutic effect

Biochemical pathways in cells are seldom isolated. Fully inhibiting one enzyme may deplete a specific product or stress the cell but will rarely shutdown an entire pathway in vivo. Other intersecting metabolic routes can assist in replenishing products and may even completely overcome the induced ‘roadblock’ [83-87]. Additionally, being tiny crucibles of strained genetic evolution, cancerous tissue is constantly battling to survive utilizing the sheer brute force of mathematical chance. Cancer cells require only a single chance adaption to a drug to gain resistance [88,89]. The method of combination therapy seeks to address these problems, since a tumor cell is far less likely to gain resistance to multiple drugs before dying. Further, different drugs can act to inhibit alternate pathways or sensitize the cell to other drugs, leading to synergistic effects. Fertig et al. designed an anti-CD20 antibody, a humanized B-Ly1 antibody, to be used alone and in combination with several proteasome inhibitors, including Bortezomib, 376

rituximab and cyclophosphamide [90]. Mice xenographs of SU-DHL-4 non-Hodgkin’s lymphoma cells showed 87% inhibition with the antibody alone [90]. Although this therapy was effective by itself, combination therapy with bortezomib resulted in lymphoma regression and complete tumor remission with no regrowth in four of nine animals [90]. Forster capitalized on patenting the usage of several types of glucocorticoids in conjunction with proteasome inhibitors to mitigate brain or spinal cord damage following an ischemic event [91]. By stabilizing the blood--brain barrier with glucocorticoids to diminish tissue edema and administering a proteasome inhibitor in an attempt to prevent the degradation of the inhibitory protein IkB-a, which downregulates NF-kB, a necrosis factor, neuronal death can be abated long enough until the tissue can be reperfused or oxygenated [91]. Delanzomib (Figure 1-8) is another compound discovered some time ago with a similar pharmacophore as bortezomib [92]. This drug has recently gone through Phase I clinical trials for treatment of MM and advanced solid tumors [93]. Delanzomib does appear to alleviate some of the peripheral neuropathy seen in bortezomib treatments but it still suffers from many of the same side effects, albeit to a lesser degree [93]. Although delanzomib is currently being researched as a possible cancer treatment, Ruggeri and Seavey have already submitted a patent for its use as a potential treatment of lupus [94]. Studies have found that the administration of delanzomib improved survival and reduced lupus nephritis in MRL/lpr and BWF1 mouse models [95]. Some patents mentioned previously that contained additional outlines for possible combination therapies are 5AHQ (Figure 4-3), which when combined with 10 nM concentration of bortezomib exhibited a synergistic effect across multiple cell lines, including MM, leukemia and solid tumors [65]. The T-L inhibitor, NC-022 (Figure 2-4), developed by Kisselev et al. exhibited a strong synergistic effect with other CT-L inhibitors [40]. When combined with 33 nM bortezomib, an 81 ± 19% growth inhibition of MM cells compared to 47 ± 13% with bortezomib alone at the same concentration was noticed [40]. The authors also tested the compound with a minor 3.7 nM concentration of carfilzomib to achieve a 64 ± 15% growth inhibition in MM cells, a substantial contrast to the near negligible 1 ± 9% growth inhibition with the same concentration of carfilzomib alone [40]. A patent can be obtained solely on a method of using a certain chemical entity. However, if there exists another patent on the structure of that chemical entity, the applicant of the method--of-use patent would need the permission of the other patent holder to be able to legally practice the method-of-use invention. When this occurs, the other patent is said to ‘dominate’ the first patent. With this in mind, patents that solely cover the methods of using a combination of chemical entities tend to be lower on patent hierarchy. Although easily overshadowed, the value of a pure method of use patent becomes apparent if no one can obtain a patent




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Figure 6. Unique chemical designs are shown.

for the chemical entity itself. This can manifest when a chemical entity is found to occur in nature. Although they can be discovered or isolated, natural products themselves are not patentable subject material. The caveat lies in that distinct methods of using natural products are patentable subject material. In this case, there is more of a level playing field, where a chemical entity structure patent cannot dominate a method of use patent. 4.

Expert opinion

In general, a person has a right to patent an invention in USA as long as the invention is not an abstract idea, a law of nature, or naturally occurring, and is useful, novel and non-obvious to one of ordinary skill in the art. What that equates to in layman’s terms is that an invention must be conceived of and made by humans, must have some useful purpose, must have never been known or publicly described before seeking a patent and is not an obvious invention to one with general and basic skill and knowledge in the field. Many factors come into play when deciding to file a patent application for a particular invention, with the potential value of the

invention’s utility often being a major factor. In the pharmaceutical field, one can think of a chemical entity’s value as hierarchical; patents on the structure of new chemical entities are often considered the most valuable, because they carry the most weight in terms of excluding others from making or practicing a patented invention. No one else can use that chemical entity for any purpose without the holder’s permission throughout the duration of the patent term, because they own the rights to make and use the structural matter. The fact that most of the patents and applications that we uncovered in our searches include, in some way, claiming new chemical entities themselves is reflective of the prime value of this type of patent position. There are over 10 structurally distinct classes of proteasome inhibitors, signifying a rich diversity of chemical space. This is important for the novelty of a patent; however, nearly all of the aforementioned chemical entities can be categorized into two general types based on the mechanism of action: a chemical warhead affixed to peptide for recognition or copying EGCG to make a competitive inhibitor. Most patents make use of a chemical warhead and a peptidomimetic moiety to selectively bind to the CT-L active site, ignoring the other


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two sites completely, and covalently attack the catalytic threonine. Covalent inhibitors are chemically reactive, tending to produce toxic metabolites and have severe off-target reactions. As a result, competitive inhibitors may be more desirable when tissue targeting is not considered, since current competitive inhibitors tend to have less severe side effects. This trend is not absolute, however, as the mechanisms of competitive inhibitors are often less understood compared to noncompetitive inhibitors, possess poorer potency and many have a similar number of off-target interactions. Based on the patent landscape, research into competitive proteasome inhibitors is limited and innovation in this field is also something to be desired. Repeatedly, others attempting to make a true competitive inhibitor synthesize EGCG analogs based on the information of EGCG--proteasome b5 interactions. The bioavailability of EGCG and analogs poses pharmacological challenges but provides a unique role in the realm of prevention vis-a`-vis diet for this class of compounds. This argument of novelty is semantic but it does attempt to expose entrenched methods of drug design. Although the practice of improving a previously discovered pharmacophore does yield results, it is essential to focus on developing new mechanisms of action and, more importantly, specific targeting. Specific targeting of cancer cells has remained an elusive goal. Due to this, most drugs attempt to take advantage of the fact that oncogenic tissues tend to be more susceptible to cytotoxic effects than normal healthy tissue. Therefore, introducing compounds that cause global toxicity should, theoretically, kill the cancer before killing the patient. This concept has yielded less than desirable results and has often led to the question of which is worse, the cure or the disease. Current attempts at proteasome inhibitors are no different. Whereas global inhibition of the proteasome has been shown to have limited toxic effects on healthy tissue, this does not render healthy tissue immune to proteasome inhibition or resistant to other off-target effects that may arise due to the presence of a chemically reactive warhead. Further, bortezomib might have some off-targets, which are associated with the emergence of peripheral neuropathy. There has been evidence in clinical trials that bortezomib may have other proteasome-independent mechanisms of action in addition to proteasome inhibition [96-103]. These articles show alternate pathways being activated or downregulated, leading to cytotoxicity and possible causes of adverse drug effects. Analysis of gene expression profiles and single nucleotide polymorphisms (SNPs) of myeloma plasma cells and peripheral blood samples have identified genes and SNPs associated with mitochondrial dysfunction and DNA repair [98]. Whereas these can be expressed through proteasomal pathways, interference with ribosome function, essential DNA damage pathways and dysregulation of many other metabolic processes can be attributed to other off-target interactions [104,105]. Previous studies have indicated that bortezomib-induced neurodegeneration in vitro occurs exclusively by way of a proteasomeindependent mechanism and that bortezomib inhibits several 378

non-proteasomal peptidase targets in vitro and in vivo [106,107]. Another gene expression profiling study found that the cytotoxic effects of bortezomib were not p27-dependent and confirmed that bortezomib increases the expression of genes associated with endoplasmic reticulum stress, whereas proteasome subunit knockdown alone does not [108]. Consistently, carfizolmib, which is more specific to inhibit the CT activity than bortezomib, has been shown to have decreased levels of peripheral neuropathy in myeloma patients, compared to bortezomib. Along this line, any compound utilizing a chemical warhead will suffer from these off-target interactions to some extent. Although a peptide mimic attached to the chemical warhead increases selectivity, the reactivity of these warheads is independent of binding with intended targets and it is likely that the warhead will react with other targets in similar chemical environments. If a drug can properly protect the warhead until binding, global inhibition is still a viable strategy so long as the side effects can be suitably mitigated. On the other hand, toxic compounds can be utilized with great efficacy if they can be delivered exclusively to cancer cells. Either way, any truly effective cancer therapy must involve a solution for specific targeting and/or exclusive delivery to cancer tissue. Over the past decade, proteasome inhibition has emerged as an effective method for treating MM and other blood cancers, in which bortezomib plays an important role. However, some limitations were identified in bortezolmib’s use. A prominent factor is adduced from the severe side effects that relegate clinical studies of bortezomib to late stage, terminal cancer patients. In late-stage cancer, treatments utilizing proteasome inhibition are ineffective, since apoptotic machinery in the cancer cell is usually dismantled. Carfilzomib has improved specificity and decreased peripheral neuropathy. It will remain to be further investigated if carfilzomib can assuage some criticisms of proteasome inhibition as a cancer therapy in practical application. Hopefully, researchers can learn from the difficulties of bortezomib and carfilzomib and develop other clinical candidates and therapeutics that can attain the perfect balance of potency, toxicity and bioavailability in the future.

Acknowledgments The authors would like to thank N Shakfeh for her assistance in drawing the chemical structures. R Metcalf and LM Scott have contributed equally to the manuscript.

Declaration of interest This work was supported by the Moffitt Cancer Center Research Institute, the University of South Florida College of Arts and Sciences, Karmanos Cancer Institute and Wayne State University. The authors declare no conflict of interest and have received no payment in preparation of this manuscript.



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Affiliation Rainer Metcalf1, Latanya M Scott2, Kenyon G Daniel*1,4 & Q Ping Dou†3,4 †,* Authors for correspondence 1 Moffitt Cancer Center, Chemical Biology Core, 12902 Magnolia Dr SRB3, Tampa, FL 33612, USA 2 Moffitt Cancer Center, Office of Technology Management and Commercialization, 12902 Magnolia Dr MRC-TTO Tampa, FL 33612, USA 3 Wayne State University, Hudson Webber Cancer Research Center, The Developmental Therapeutics Program, 4100 John R. Street Room 540.1, Detroit, MI 48201, USA Tel: +1 813 745 5734; E-mail: [email protected] 4 Wayne State University, Karmanos Cancer Institute, 540.1 HWCRC, 4100 John R Road, Detroit, MI 48201, USA Tel: +1 313 576 8301; Fax: +1 313 576 8307; E-mail: [email protected]

382


Acyltransferase inhibitors: a patent review (2010-present).

The resistance mechanisms of proteasome inhibitor bortezomib.

An oral proteasome inhibitor for multiple myeloma.

Ubiquitinated proteasome inhibitor is a component of the 26 S proteasome complex.

Upregulation of proteasome activity rescues cardiomyocytes following pulse treatment with a proteasome inhibitor.

Gene patents.

AZT patents.

Piecemeal patents.

Epidermal growth factor receptor inhibitors: a patent review (2010 - present).

Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity.

Proteasome inhibitor carfilzomib complements ibrutinib's action in chronic lymphocytic leukemia.

Oral proteasome inhibitor with strong preclinical efficacy in myeloma models.

Prospective iterative trial of proteasome inhibitor-based desensitization.

A potent and selective inhibitor for the UBLCP1 proteasome phosphatase.

Melatonin as a proteasome inhibitor. Is there any clinical evidence?

Proteasome inhibitor carfilzomib interacts synergistically with histone deacetylase inhibitor vorinostat in Jurkat T-leukemia cells.

Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma.

Product patents.

Patents and literature.

Patents for genomes?

Patents, injunctions, and sutures.

Prophetic patents in biotechnology.

Aurora kinase inhibitor patents and agents in clinical testing: an update (2011 - 2013).

The novel β2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.