De Novo Design of a LOXL2 Mini-Protein Inhibitor to Slow Metastasis

PAPER & RESEARCH BY: ISABELLE S. Y. COBURN MENTORED By: DR. ERIKA DE BENDEDICTIS NOVEMBER 12, 2020

ABSTRACT

The process of invasion and metastatic spread of cancer cells involves the restructuring of the extracellular matrix (ECM) into a premetastatic niche. This is ultimately accomplished by Lysyl Oxidase (LOX) enzymatic catalysts which restructure the ECM with a scaffold that causes collagen to become dense, straightened, cross-linked, and rigid compared to a pre-cancerous ECM, forming a microchannel for primary cancer tumor cells to escape the primary tumor environment enabling invasion and metastasis. In malignant tumor microenvironments, Lysyl Oxidases perform a crucial role in enabling a mechanotransduction circular feedback loop that ultimately promotes invasion and metastasis. Lysyl Oxidase Like-2 is a vital target for inhibition to disrupt invasion and metastasis. It has been explored with small-molecule therapeutics and other protein inhibitors, some successful albeit with disqualifying side-effects. Some protein inhibitors are currently in trials, and I do not yet know their efficacy. For a therapeutic to be successful, it must be highly targeted to malignant tumor sites, have minimal toxicity, be effective in inhibiting LOXL2 disrupting collagen-1 crosslinking, and be reversible once treatment has succeeded. A highly targeted approach using gold nanoparticles coupled with the benefits of using simple mini-protein LOXL2 inhibitors could be a highly successful systematic approach that achieves all of these objectives. Using de novo computational protein docking and design, I analyzed and selected some mini-protein LOXL2 inhibitor candidates. Using known gold nanoparticle structures with proven targeting moieties, I propose a delivery mechanism for delivering mini-protein 5UP5 and 5UYO as LOXL2 inhibitor candidates that could successfully disrupt collagen-1 cross-linking and lull activated CAF’s into quiescence and halt invasion and metastasis.

KEYWORDS: Extracellular Matrix (ECM), collagen 1, cross-linking, cancer-associated fibroblasts, Lysyl Oxidase Like-2, LOXL2, Gold Nanoparticle, Pre-metastatic niche, de novo protein design, mini-protein inhibitors

INTRODUCTION

Cancer is primarily caused by mutations or alterations in the expression of proto-oncogenes, tumor suppressor genes, and other aspects of DNA repair. Only 5-10% of cancer deaths are caused by inherited genetics. Most cancer deaths are a direct result of environmental factors (i.e., exposure to radiation or pollutants) and also unhealthy lifestyles, which include poor diet, lack of activity, tobacco usage, and stress (1, 2). 40% of people may be diagnosed with cancer during their lifetime, and cancer is considered to be currently one of the leading causes of death worldwide (3). 90% of cancer-related deaths are due to the spread of cancer from a primary tumor site to a more critical site such as the bone, brain, liver, and lung (4). Cancer metastasis is promoted due to the extracellular matrix (ECM) remodeling to a pre-metastatic niche, one that enables cancer cells to invade and leave the primary tumor micro-environment. This process of remodeling the ECM of previously healthy tissue to one that becomes diseased and labeled as cancerous stroma is one that kicks off with activated, cancer modified, overactive cancer- associated fibroblasts (CAFs) (5). This results from a long chain of events starting with CAFs releasing matrix metalloproteinases (MMPs) that degrade the ECM. Simultaneously, collagens and other materials are secreted into the ECM that is used in rearranging the ECM into a pre-metastatic niche (6). This rearrangement includes straightening or linearizing the collagen fibers and arranging them in parallel patterns instead of curving crosslinking patterns. The fibrils align in the ECM perpendicular to the tumor’s boundary providing tracks that guide cancer cells’ migration (7). Collagens are also produced in much greater numbers than would typically be found in non-cancerous tissue (8). As a result of the accumulation, linearization, and straightening and condensed arrangement of the highly cross-linked collagen fibers and elastin, the ECM becomes stiffened. In normal tissue, collagen fibrils are relaxed and non-oriented. In cancer tissue, they align perpendicular to the tumor boundary, associated with malignant metastatic progression (9, 10). The non-random orientation of ECM fibers is called anisotropy. It forms a concentrated gradient capable of generating differential tension and, in turn, migration micro-tracks that assist in guiding malignant cell migration (11). This stiffening of the stroma sends mechanical cues to the cancer cells themselves, making them elongated and sending mechanical signals to break off from the primary tumor and begin a journey enabled by the rearrangement of the ECM, eventually breaking through a leaky basement membrane into the vascular or lymphatic system (12). This is how the metastatic spread of cancer outside of the primary tumor begins. Most conventional cancer treatments, whether they have been radiological or chemical, have been directed at cancer tumors themselves. Cancer tumors typically become deadly due to metastasis (13). Metastasis is accomplished through a complicated process with multiple steps: 1. loss of cell-to-cell adhesion 2. loss of cell-to-

matrix adhesion 3. Epithelial to mesenchymal transition (EMT), 4. Intravasation 5. Ability to survive in circulation 6. Vascular arrest 7. Extravasation 8. an ability to establish metastases at a distant site (14-16). Often tumors become more challenging to treat with conventional treatments due to the ECM restructuring, enabling the invasion and metastasis. This process involves the overexpression of several proteins, which ultimately disrupts the development of anticancer therapies (3). With the combined deadly nature of metastasis and difficulty of conventional cancer therapeutics being useful in a restructured ECM, I believe that it is ideal for targeting the ECM restructuring that enables invasion and metastasis.

BACKGROUND

Cancer cells along with activated CAFs drive the process of invasion and metastasis by releasing collagens, various enzymes that include both matrix-metalloproteinases (MMP’s), Lysol Oxidases (LOX), and a variety of other structural ECM proteins, which all together act in a concerted cycle which degrades then rebuilds the surrounding ECM turning it into remodeled cancer stroma (17, 18). This is accomplished primarily as part of the ECM remodeling process and is mainly due to fibroblasts being activated into CAFs. This starts a vicious cycle of restructuring the collagen structure within the interstitial ECM with a higher concentration than normal concentration of MMPs, collagen-1 fibers, and LOXs, which lead to excessive collagen-1 fiber and elastin cross- linking. This cross-linking creates a scaffold with heightened tension strength, transforming the ECM into a structurally stiffened fibrous cancer stroma (9). This strengthened structure through mechanotransduction signals to activated CAFs to promote continued and increased production of MMPs that degrade the existing interstitial ECM and increase collagen and LOXL enzyme secretion, all in an endless collagen-1 cross-linking circular loop

(19-21). This mechanotransduction process is part of a circular signaling process that impacts TGF-Beta, VEGF,

MMPs, and LOXL2 production (22, 23). This structure ultimately enables invasion and metastasis because it leads to an alignment and straightening of the collagen fibrils to create micro tracks and enables and promotes EMT and angiogenesis, further pushing the primary tumor to become metastatic (24, 25, 11). Early on, it might only impact the primary tumor site, but metastasis is also understood to promote conditions that enable cancer spreading from the primary tumor site to a secondary site via the “seed and soil hypothesis” (4). This restructuring of the interstitial ECM creates a tumor microenvironment that hinders the effectiveness of conventional cancer treatments. Once cancer tumors reach dimensions in excess of >1cm3, hypoxic regions are formed, which generally makes them more resistant to anticancer chemotherapy and radiotherapy. Hypoxia also has been shown to predispose tumors to increased invasion and metastatic processes

(26). This is due to creating a “web” of protective material around the primary tumor that significantly hinders the effectiveness of treatments like Radiotherapy - Knockdown of LOXL2 in CRPC cells has been shown to enhance cellular radiosensitivity substantially. This is the primary mechanism by which radiosensitization increases may be attributed to the EMT process’s reversal associated with LOXL2 knockdown (27). Chemotherapy’s effectiveness often proves challenging if not disappointing because drugs cannot reach close enough to the malignant cancer cells to have much effect on them (28). In general, a dense, firm extracellular matrix around a tumor hinders therapies that need to be delivered directly to cancer tumor cells to be most effective.

LYSYL OXIDASE LIKE-2

One attractive candidate to accomplish this would be the inhibition of Lysyl Oxidase Like-2 (LOXL2). LOXL2 is a member of the LOX family. LOXL2 is a secreted copper-dependent amine oxidase, and its main enzymatic role is to catalyze the oxidation of lysine, resulting in the formation of peptidyl alpha-aminoadipic- semialdehyde, which spontaneously condenses to form covalent cross-links between collagen-1 fibrils as well as between collagen-1 fibrils and elastin. From this, the stabilizing, strengthening, and “firming” of the ECM is accomplished. This is accomplished by the oxidative deamination of peptidyl lysine residues through the terminal E-amine of hydroxylysine or lysine residues found in the telopeptide regions of either collagen or elastin and generates a resultant allysine (29-32). LOXL2 has a highly conserved carboxyl (C) terminal domain that contains a copper-binding mechanism, lysine tyrosyl quinone (LTQ) residues, and a cytokine receptor-like (CRL) domain, all of which proves essential for enzymatic catalytic activity with collagen fibers (33).

However, some prior studies show that LOXL2 may act as a tumor suppressor (34). It appears paradoxical where there are at least instances when LOXL2 has a tumor suppression role, but at later stages of cancer, it serves to be invasion and metastasis enabling. LOXL2 has been demonstrated to contribute significantly to the processes of EMT, senescence, cell-adhesion, cell-migration, and ultimately invasion and metastasis (35-38). LOXL2, however, is for certain responsible for the cross-linking, leading to stiffening and straightening of collagen fibers triggering the mechanotransduction cycle with activated CAFs promoting continued ECM remodeling, leading to metastasis. LOXL 2 has been discovered to have a significant role in the activation of CAFs. Several studies have demonstrated that SRC and FAK can be activated through LOX-mediated increases in collagen cross-linking and the resultant matrix stiffening through stimulated downstream FAK and SRC transmembrane integrin activation (39,40).

LOXL2 has been demonstrated to increase the expression of Vascular Endothelial Growth Factor

(VEGF), which is a signaling pathway that plays a vital role in angiogenesis within endothelial cells (41-43). Overexpression of LOXL2 has also been shown to increase HIF-1Alpha expression under hypoxic conditions, which leads us to believe that LOXL2 and HIF-1Alpha reinforce each other in the promotion of tumor progression (44). In addition, LOXL2, in part, regulates H2O2 production, by a mechanism of the enzymatic reaction between collagen1 and LOXL2, as a by-product of that reaction. H2O2 is known to activate a signaling pathway of PI13K/Akt, which then increases HIF-1Alpha protein synthesis, showing that LOXL2 production creates a positive feedback loop that promotes the proliferation of tumors (45). This process of producing excessive amounts of LOXL2 is known to occur in activated CAFs. By inhibiting LOXL2, its cycle of production would be slowed down or possibly even stopped. It has been demonstrated that upregulation of LOXL2 highly impacts ECM stiffness, which occurs as a direct result of activation of integrin ALPHA5/B1/JNK/c-JUN signaling pathways, leading to the secretion of LOXL 2, Matrix metalloproteinase-9 (MMP-9), and Stromal cell-derived factor1 (CXCL12) all of which contribute to the stromal pre-metastatic niche formation (46). This is primarily accomplished through Cell-ECM interactions that involve reciprocal forces that are predominantly mediated and translated into intracellular signals through integrins and contribute to the mechanotransduction and regulation of LOX with different mechanical forces (11). LOX expression is dependent on the physical properties of the cellular microenvironment. This includes the interaction of integrin Alpha2Beta1 with collagen-1 when combined with LOX as this induces stabilizing collagen and elastin cross-linking, forming integrin complexes, which lead to the activation of the TGF-beta pathway, which then again promotes and increases LOX production (47, 48). Recognition of this feedback loop is incredibly essential, where increased ECM stiffness results from pathological situations where malignant tumor progression may be occurring (49). We know that LOXL2 acts as a catalyst linking collagen-1 fibers together by a copper cofactor. From a biochemical perspective, copper is a cofactor for the LOXL2 enzyme and a determinant of its activity in connective tissues. Lysyl oxidase catalyzes post-translational oxidation of residues lysine and hydroxylysine. The peptidyl aldehydes formed are active centers for the cross-link formations for both collagen and elastin. It requires oxygen as the recipient of electrons from the substrate. The products of the reaction are NH3, H2O2and a peptidyl derivative of hydroxylysine or lysine (hydroxylysine or allysine) (50). By eliminating LOXL2, the biochemical mechanism would mainly disable the cross-linking and soften the overall interstitial extracellular matrix. By weakening and softening the ECM, it would disable the mechanotransduction necessary to activate CAFs and, in turn, disrupt the metastatic process from continuing. This should have the impact of stopping the

mechanotransduction signaling to produce TGF-Beta, which in turn would have the impact of deactivating or putting the CAFs into quiescence. Under normal conditions, TGF-Beta and LOXs must be tightly maintained in the ECM to maintain homeostasis. In cancer, neoplastic cells typically evade the regulatory scheme and end up producing LOX overabundantly. As a result,t TGF-Beta becomes overexpressed and dysregulated in cancer, and it is thought that the increase in LOXs is probably, at least in part, TGF-Beta driven. Under normal homeostatic conditions, TGF-Beta increases the expression of LOX, and LOX, in turn, acts as negative feedback to then downregulate TGF-Beta (51, 52). LOX enzymatic activity, instead of its byproduct H2O2 has been shown to be responsible for suppressing TGF-Beta signaling because the TGF-Beta molecule is targeted at the basic lysine-rich

C terminus and is inactivated by oxidative deamination of lysine residues (53, 54). LOX catalyst activity has a couple of different methods that can influence TGF-Beta degradation, whether by oxidative deamination at the C terminus of TGF-Beta or indirectly through the activation of HTRA1, which is a secreted protease capable of degrading TGF-beta (55-57). This, in turn, basically disrupts the cycle of production of LOX, MMPs, VEGF, etc., which promote the metastatic restructure of the interstitial ECM. It is also thought that LOXL2 enables the “seed and soil theory” and helps plant tumor cells from the primary tumor microenvironment to secondary tumor sites. By reducing LOXL2, it will also decrease the ability of cancer even if escaping the primary tumor site from “planting” itself into a secondary tumor site. It has been demonstrated that inhibiting LOXL2 makes the seeding of escaping cancer cells from the primary tumor microenvironment extremely difficult to spread to a secondary site because, at any possible secondary sites, the “soil isn’t fertile.” LOX enzymes have been shown to not only modulate the primary tumor ECM, but also have a role in modulating the ECM of distant organs to form the pre-metastatic niche even before the arrival of malignant tumor cells (58). Unfortunately, currently, the mechanisms behind this pre-metastatic niche formation are not fully understood. It is still under investigation whether tumor-derived factors circulate in the body and exert effects on ECM remodeling or particular tumor secreted factors that initiate the reactions and signaling events in specific tissues. Either way, it has a role, whether the cause or effect in this process (59). LOXL2 inhibition has also been shown to inhibit the production of MMP2 and MMP9, which are the primary enzymes that degrade collagen IV, which is the primary collagen structure of the basement membrane, which have to be breached in order for cancer cells to escape the primary tumor microenvironment (60). If the basement membrane can be prevented from being breached or the breach can be slowed, cancer cells could be contained in the primary tumor microenvironment. In essence, this could prevent or correct a leaky basement membrane, making it much more difficult for cancer cells from the primary tumor site to enter the vascular or lymph systems.

KNOWN CURRENT LOXL 2 INHIBITORS There are currently a few different methods of inhibiting LOXL2. First, there is a well-known LOXL2 inhibitor called BAPN, which has proven highly effective in inhibiting LOXL2. However, BAPN is a mechanism-based, irreversible inhibitor which is toxic outside of therapeutic purpose in fibrotic diseases (61).

There are also several other early simple LOXL2 inhibitors including D-penicillamine (62), Thiram (62), Disulfiram

(62), Benzylamines (63), Alkyldiamines (64), Allylamines (65, 66), Homocysteine thiolactone (67), Isoniazid (68), Aryl

Hydrazines (68), Trans-2-phenylcyclopropylamine (TCP) (69), and N-(5-aminophenyl)aziridine (70). They’re extremely simple structures with little possible sites for chemical modification that could improve the many different disqualifying side-effects resulting from these inhibitors. B-Aminopropionitrile (BAPN), a standard method of irreversible LOX inhibition, works by targeting the active covalent bonding site of LOX (71, 61, 72). Unfortunately, BAPN never proved to work very well in clinical treatments due to variable potency and several known disqualifying side-effects (73-75). Another LOXL2 inhibitor made by Gilead called Simtuzumab (AB0024), which is the humanized version of the antibody, went into clinical trials but failed (76, 77). Its failure was attributed to a lack of tissue-specific targeting as well as a lack of clear dosing methodology (78, 79). Another LOXL2 inhibitor made by Pharmakea (PAT-1251) has demonstrated efficacy and has recently entered into clinical development (76, 77). Pharmaxis also developed other LOXL2 inhibitors (PXS-4878A) and (PXS-5153A), both currently in clinical trials, and both were designed for the treatment of fibrosis (80, 81). A dual LOX/LOXL2 inhibitor called PXS-S1, which was also developed by

Pharmaxis, is also in preclinical development and demonstrates some potential (82).

TARGETED DELIVERY MOTIF REQUIRED As mentioned, because of the toxicity and disqualifying adverse side-effects involved in inhibiting LOXL2, it only makes sense to do so in a targeted and contained way. Like most small-molecule anticancer drugs, there are many limitations with adverse side effects, rapid elimination from the body, non-specific cytotoxicity, multi-drug resistance, and low bioavailability (83). This is why LOXL2 inhibition can only really prove effective if delivered in a highly targeted and controlled manner. The strategy should ideally only enable the inhibition of LOXL2 within targeted active malignant tumor sites as close in proximity as possible to the CAFs that produce LOXL2. One way to do this is by using gold nanoparticles, which have many benefits as a delivery motif. They have also been shown to act as a CAF therapeutic.

First, nanoparticles through the enhanced permeability and retention effect have been shown to effectively accumulate in tumor tissues, even without any targeting ligands (84). This happens due to defective tumor vasculature, which has irregular epithelium, decreased lymphatic drainage levels, and reduced uptake of interstitial fluid, which favors retention of nanoparticles within malignant tumors (84). Gold has been long believed to have medicinal properties shown in ancient Arabian, Chinese, and Indian papers, although why it was believed to work is unknown, other than we do know that gold is one of the least reactive chemical elements, which seemingly makes it safer for medical usage (85). However, there are concerns about inflammatory response or the potential accumulation of metal in an organism as they have not yet been used enough in practice or tested long enough in in-vivo studies (86, 87). Gold nanoparticles between the size of 10-110 nm are the best size range for targeting cancer tumor microenvironments. It has been shown that nanoparticles in this size range with a neutral charge can penetrate throughout large tumors and have proven highly effective when combined with targeting moieties (88). Interestingly, it has been shown that the maximum uptake of gold nanoparticles occurs with 50 nm diameter spheres (89-91). It is thought that this is because this size is similar to lipid-carrying proteins and viruses. Gold nanoparticle toxicity has shown to be complicated, as in vivo tests have had somewhat conflicting results. It has been demonstrated that nanoparticles of sizes 3, 5, 50, and 100 nm did not induce cytotoxic effects; simultaneously, gold nanoparticles within the range of 8 to 37 nm caused severe toxicity in mice (92). Nevertheless, gold nanoparticles with approximately 50 nm are non-toxic (93). Generally, gold nanoparticles can be biocompatible, with no toxicity based on size, shape, and facile synthesis (94). Along with the size and shape of gold nanoparticles, one key attribute of gold nanoparticles is that they can be functionalized to protect aggregation, enhanced bio-compatibility, specifically targeted interactions with cells, and act as a transporter to accumulate in desired locations. For instance, surface modifications can potentially hide gold nanoparticles, preventing their removal by the mononuclear phagocytic system (MPS), also called the reticuloendothelial system (RES) (95). Functionalization is accomplished either through physical absorption or covalent attachment of ligands on the surface of the nanoparticles, and this can be achieved through using thiol linkages. The most commonly used compound used for gold nanoparticle functionalization is poly (ethylene glycol) (PEG), bound covalently to the surface atoms of the gold nanoparticles. PEG has shown promise enhancing the biocompatibility of various nanoparticles by prolonging their blood half-life (96, 97). Several studies have not found cytotoxic activity of gold nanoshells coated with PEG when introduced to human cancer cells (98, 99). It also has shown enhanced accumulation of the gold nanoshells at the tumor site (100).

Most importantly, it has been shown that gold nanoparticles that are approximately 20 nm in size have been shown to disrupt CAF fibroblasts. It has been demonstrated that 20 nm gold nanoparticles block signaling pathways and quell activated CAF’s back into quiescence. (101). Although gold nanoparticles of 20 nm size are toxic to mice, it is not harmful in humans, and further in vivo studies will need to be conducted (92). Gold nanoparticles have been demonstrated to have several other self-therapeutic values, including inhibition of mitogen-activated protein kinase (MAPK)- activation as well as epithelial-mesenchymal transition (EMT) by downregulating heparin-binding growth factors (102, 103). Lastly, gold nanoparticles have been shown to disrupt the bidirectional crosstalk between cancer cells and CAFs, essentially reprogramming the tumor microenvironment, impeding tumor growth (104). Gold nanoparticles have been shown to successfully interfere with and alter fibroblast activation levels (105). Disrupting the link between CAFs and cancer cells has great potential by either eliminating or reprogramming CAFs, inducing responses to CAF antigens, and blocking secreted CAF factors, and inhibiting cancer cell receptors (106, 107). To enable the targeting abilities of gold nanoparticles, they can be coated with antibodies that allow them to target cancer tumors and, with the size at 20 nm, can easily penetrate the leaky vascular that exists in a tumor microenvironment (105). I further propose to coat gold nanoparticles to specifically target CAF markers, which can include fibroblast activated proteins (FAP), alpha-smooth muscle actin (ALPHA SMA), or (PDGFR-Alpha), or GPR77, which has been hypothesized to be an exact CAF-targeted strategy; positioning near CAF which is ideal to both lull CAF’s into quicensance and equally an excellent strategic location to inhibit LOXL2 disabling crosslinking of collagen during the remodeling of the ECM (108, 109).

PRIOR LIMITATIONS ON DEVELOPMENT OF LOXL2 PROTEIN INHIBITORS One significant limitation that has existed until recently is that there are no NMR or X-ray structures of Lysyl Oxidase 2. Progress has been made recently when in 2019, a three-dimensional model of Human Lysyl Oxidase Like-2 was published. Only recently, a usable crystalline structure of the human LOX enzymatic protein has been available for use in computational modeling. This structure will hopefully allow for a better understanding of LOXL protein enzymatic activity (110). Although the molecular mechanisms that determine how copper is delivered to LOX enzymes are still not fully understood, it is generally agreed that copper plays a significant role in enabling LOX catalytic activity (111, 112). Human Lysyl Oxidase is a synthesized proenzyme consisting of 396 amino acid residues processed by bone morphogenetic protein-1 (BMP-1) and mammalian tolloids. These enzymes release an active form of LOX with an N-terminal propeptide (147 residues). The propeptide is extended and flexible and has demonstrated to interact with 34 different partners. LOX must exit

the endoplasmic reticulum and plays biologic roles that include a ras rescission gene activity, which puts it in the category of a matricryptin (50, 113, 114). LOX has five disulfide bridges (115), a copper ion, and a lysine tyrosyl quinone cofactor (116, 117), which catalyzes automatically when in the presence of a copper ion (118) and this cross- links the residues K320 and Y355 in the human enzyme. Inhibitors of LOX enzymatic proteins so far have proven mostly ineffective for a lot of different possible reasons, some understood, others not so well. First of all, it has only been very recently that the crystalline model of LOX catalytic protein enzymes has been available (119, 120), which severely limited the approaches available for modeling and development and only now makes possible effective structure-based design (110). Second, it has been proven that it is not easy to purify recombinant LOX proteins, as they have very poor solubility and quite often require refolding, making them incredibly difficult to work with (121). Third, recombinant LOX catalytic protein enzymes need exceptionally high concentrations of urea to be solubilized, and this severely limits the ability to screen and identify potential new effective compound opportunities. Now having a documented crystalline structure along with recent purification of active LOXL2 protein has proven to enable the development of much more advanced and hopefully effective future inhibitors (110, 121, 122).

CURRENT OPPORTUNITY FOR LOXL PROTEIN INHIBITOR De novo protein design has a lot of potential for developing small, stable, customizable proteins for targeted therapeutics. Mini-proteins are incredibly stable and are not rendered inactive with exposure to high temperatures. Probably most important, though, is that mini-protein designs typically don’t elicit an immune response. Mini-proteins can be defined as short proteins of less than or equal to 40 amino acids with well-defined folds consisting of two or more secondary structure elements, sequestered hydrophobic cores, and cooperative folding (123, 124). As such, small binding proteins potentially can be effective somewhere between monoclonal antibodies and small molecule drugs (127-129). As such, mini-proteins are a prime candidate to act as an effective LOXL2 inhibitor. Mini-proteins make sense as a LOXL2 inhibitor because the small size of mini proteins means that they have smaller hydrophobic cores and fewer non-covalent interactions when compared to most other proteins. This enables mini-proteins to be stabilized by metal binding or covalent cross-linking. There are many other advantages of using mini-proteins for LOXL2 inhibition as mini-proteins have a distinct advantage over other more complex protein structures. The entropic cost of folding protein chains is often outweighed by the enthalpy of forming these interactions, which influences their potential instability. Mini-proteins, in turn, are more stable because complexity is reduced with less folding promoting more stability. (125,126)

METHODS AND RESULTS

LOXL2 Mini-protein Inhibitor Selection Different tools and resources were used to test and analyze the efficacy of mini-proteins as LOXL2 inhibitors through computational methods and design. Crystalline protein structures were pulled from a databank to be run through a docking supercomputer to predict where mini-proteins would bind onto LOXL2. These predictions were then interpreted over an open-source visualization tool. I determined the best candidate for potential mini-protein scaffolds by calculating the solvent-accessible surface area (SASA) of a mini-protein bound to LOXL2. SASA determines the best mini-protein candidate by determining what protein has the best geometric fit onto LOXL2, allowing it to bind more effectively and easily. To start with, a three-dimensional crystalline structural model of lysyl oxidase was published and made available on May 14, 2019 (50). Without this, there wouldn't even be a starting point to target inhibition of LOXL2. With that said, LOXL2 starts in a pre-cursor state absent of the copper ion required to catalyze covalent collagen or elastin cross-linking. Ideally, the objective would be for the mini-protein inhibitor to scaffold over the area of LOXL2 that catalyzes collagen cross-linking so that it can most effectively disable the catalytic function of LOXL2. This, however, is incredibly difficult if not nearly impossible at this point to computationally model as to how this catalyst mechanism naturally occurs is not yet fully understood. So with this in mind, modeling is targeted at simply identifying mini-proteins with a high affinity of attachment and scaffolding with the LOXL2 enzyme structure.

MINI-PROTEIN LOXL2 INHIBITOR CANDIDATES I selected various structurally disparate mini-proteins where the crystalline structure was available, acquired their structures from the Protein Data Bank (RCSB PDB), and used their provided crystallized biological models to run through a protein docking algorithm. Mini-proteins can be used as inhibitors and promoters due to their small size, and many of them can bind to multiple places on larger molecules. Many properties of mini-proteins can affect their ability to bind onto other proteins, such as high binding affinity and shape complementarity. In protein design, it is good to have a diversity of scaffolds consisting of different physical structures and sizes because binding compatibility between two proteins usually cannot be foreseen until tested through computational means. Mini-protein 5UYO is a relatively small and simple molecule consisting of two main alpha- helices. Its simplicity may allow it to fit onto a protein’s surface more easily. On the other hand, 6VGB has a

much more complex structure made up of alpha-pleated and beta-pleated sheets. It takes on a more complex shape in contrast to 5UYO’s simple structure, which makes it predictably more challenging to fit onto the surface of another protein. Nevertheless, using various unique structures enables an analysis of what type of structures have the greatest likelihood of successfully scaffolding the LOXL2 enzyme. By analyzing their success through docking, it makes it possible to better zero-in on other similar structural targets that might yield greater success. MINI PROTEINS SELECTED FOR ANALYSIS

6VGB 5UOI 5UP1 5UP5

5UYO 6W90 6VGA 6VG7

Figure (1): Eight different mini-proteins selected varied in shape and size and used to dock onto LOXL2 to determine which structure would be geometrically compatible with LOXL2. These crystalline structures were selected from the RCSB PDB and viewed over PyMOL (131).

PROTEIN DOCKING - CLUSPRO With the eight mini-protein crystalline structures acquired over the PDB to dock onto LOXL2, I compared their docking with that of collagen-1 (1BKV) docked to LOXL2 using the docking tool ClusPro. ClusPro is used to analyze how protein crystalline structures are physically bound. Protein docking is done over a supercomputer that uses algorithms to predict how two protein structures will bind to one another. All eight mini-proteins were successfully docked onto LOXL2, and the results were further analyzed over PyMOL. After uploading each protein’s PDB ID into the ClusPro server, the docking algorithm on the supercomputer is then run, producing multiple models of docked possibilities for each protein pair. Ideally, a mini-protein should bind directly onto a ligand, and the chance that it may dock in different sites on a single

protein is likely for mini-proteins due to their smaller and simpler shape. This enables them to have more possibilities of where to bind onto a protein.

COLLAGEN-1 DOCKED WITH LOXL2

Figure 2: Modelled collagen-1 binding in various ways with LOXL2. Collagen is the green structure and LOXL2 is the V-shaped structure in the diagram.ClusPro provides multiple models which is why there are 10 graphic representations of the different sites of docking, which could then be further analyzed over PyMOL. Note that the docking of collagen-1 with LOXL2 is problematic, which is not expected for a ligand and receptor, which commonly bond in nature. This could be the result of the crystalline structure of LOXL2, which is a contrived model rather than a more accurate NMR or X-Ray derived structure. Additionally, how Collagen-1 and LOXL2 naturally bond and act as a catalyst is not yet fully understood. All of this might contribute to the unexpected docking results of Collagen-1 with LOXL2. None of the above representations are what would be expected in a naturally occurring catalytic reaction between Collagen-1 and the enzyme LOXL2. We know that Copper ion must be exchanged and this copper ion is located approximately in the lower part of the V-like LOXL2 structure. (131-135).

PyMOL - CALCULATING SASA + VISUAL MODELS PyMOL was the primary platform used to analyze physical models of crystallized proteins from the protein data bank, and the docked results from ClusPro. Using the PDB ID from the protein data bank, which

provided the crystallized structures of each mini-protein, I was able to view the proteins’ physical structures over PyMOL. As a visualization tool, PyMOL can also run calculations that ultimately help determine the best mini- protein scaffolding candidate through properties such as binding affinity and shape complementarity. Some proteins have high binding affinity due to large amounts of hydrophobic residues located externally on their structures, making them “sticky” and easier to attach to proteins. In my research, I calculated each protein’s buried solvent surface area (SASA), which could be directly correlated to its shape complementarity with LOXL2. The SASA calculates the interface area between a receptor and its ligand by adding the surface area of both the target and ligand together and subtracting the surface area of the complex of the two proteins together. To test for a mini proteins efficiency and its ability to bind to LOXL2, SASA is used to determine the percentage of coverage on a protein’s entire surface area that entails its interface with LOXL2. A higher percentage SASA would indicate a better geometric fit between the two docked proteins, which would prove a mini-protein to be a promising candidate to scaffold a suitable binder. SURFACE AREA OF COLLAGEN-1 & EACH MINI-PROTEIN

1BKV 6VGB 5UOI 5UP1 5UP5 5UYO 6W90 6VGA 6VG7

model ligand interface ligand interface ligand interface ligand interface ligand interface ligand interface ligand interface ligand interface ligand interface

0 5279 2660 6052 2882 3561 2220 5166 2980 3316 2074 3412 1511 7596 2623 6164 2473 5683 1536

1 5211 2703 5927 1814 3471 2064 5237 1993 3331 2090 3632 2581 7329 1718 6054 1518 5575 1805

2 5143 2692 5782 1981 3408 1153 5423 3054 3328 2162 3527 2291 7387 2208 5801 2261 5469 1699

3 5259 3606 5789 2239 3450 1686 5638 4323 3422 2304 3631 2317 7434 2114 5890 2040 5559 2300

4 5163 2939 5831 1910 3340 2189 5248 2934 3263 1801 3675 3243 7407 2127 5825 2353 5534 1480

5 5133 2002 5670 2398 3522 2180 5460 3793 3465 2046 3601 1801 7423 1992 5996 2321 5557 1792

6 5208 2110 5797 2365 3435 2412 5473 2670 3295 2036 3597 2765 7747 2851 5901 1592 5583 1833

7 5111 2504 5804 1378 3231 1609 5299 2962 3341 2445 3440 2319 7407 2127 5983 3851 5640 2252

8 4957 2805 5827 2267 3366 1649 5268 2137 3378 1898 3613 1304 7573 1995 5966 3566 5544 1926

9 5238 2706 5889 1953 3567 2126 5354 2452 3262 1494 3494 1536 7452 2518 5847 1122 5627 2300 Figure 3: The surface area of collagen-1 (1BKV) and each mini-protein as well as the area of their interface with LOXL2 for 10 different attachment sequences were calculated over PyMOL (132) in order to determine each protein’s SASA. Surface area is measured in square ångström (Ų). The surface area of the entire mini-protein was provided by PyMOL with the command ‘print ligand_area’.

PERCENTAGE OF MINI-PROTEIN BOUND ONTO LOXL2 model 1BKV 6VGB 5UOI 5UP1 5UP5 5UYO 6W90 6VGA 6VG7

0 50% 48% 62% 58% 63% 44% 35% 40% 27%

1 52% 31% 59% 38% 63% 71% 23% 25% 32%

2 52% 34% 34% 56% 65% 65% 30% 39% 31%

3 69% 39% 49% 77% 67% 64% 28% 35% 41%

4 57% 33% 66% 56% 55% 88% 29% 40% 27%

5 39% 42% 62% 69% 59% 50% 27% 39% 32%

6 41% 41% 70% 49% 62% 77% 37% 27% 33%

7 49% 24% 50% 56% 73% 67% 29% 64% 40%

8 57% 39% 49% 41% 56% 36% 26% 60% 35%

9 52% 33% 60% 46% 46% 44% 34% 19% 41%

average: 52% 36% 56% 55% 61% 61% 30% 39% 34% Figure 4: Taking the surface area of both the ligand and the interface from the table in figure (3) for each of the 10 different attachment sequences, I then calculated what percentage of the mini-protein bound onto LOXL2 from each model of each protein. These percentages were then averaged to get a better understanding of which mini-protein is the most geometrically compatible with LOXL2.

DIAGRAM OF LOXL2 WITH MINI PROTEINS 5UP5 AND 5UYO ATTACHED IN ONE OF 10 POSSIBLE LOCATIONS A B

Figure 5: Diagram of LOXL2 with mini-protein inhibitor candidates modeled as structurally attached. Based on the average SASA percentage, both 5UP5 (A) and 5UYO (B) are the most effective in binding onto LOXL2. Structural images were generated using Pymol (134).

VISUAL REPRESENTATION AND ANALYSIS OF MINI-PROTEIN 5UYO DOCKED TO LOXL2

Figure 6: Diagram of LOXL2 docked with mini-protein 5UYO inhibitor candidate modeled as structurally attached. There are a total of 10 different orientations of LOXL2 attached to to mini-protein 5UYO. It can be seen that the attachment location varies significantly but the prime candidates to be effective over the catalytic location can be seen in diagrams 1, 6, 7, whereas the other locations of attachment would probably not be effective in scaffolding the catalytic location of LOXL2 and Collagen-1. Structural images were generated using Pymol (134).

In figure 6, it denotes the 10 different attachment sequence locations of mini-protein 5UYO to LOXL2 as simulated in Pymol. Overall 5UYO had an average surface area binding of 61% which also makes it a prime candidate overall. Beyond this average surface area binding it is also important to try to have a scaffold that covers the enzymatic catalytic binding site where Collagen-1 and LOXL2 react. While the process of this reaction is not fully understood or even observed, we do know that in order for the reaction to occur their needs to be a copper ion exchange between the LOXL2 enzyme and Collagen-1 binding site. We do understand that copper is in the center of the V like structure in LOXL2. Location-wise, I can derive that 6 which has a surface area binding of 77% and 7 with a surface area binding of 67% could be effective LOXL2 inhibitors.

VISUAL REPRESENTATION AND ANALYSIS OF MINI-PROTEIN 5UP5 DOCKED TO LOXL2

Figure 7: Diagram of LOXL2 docked with mini-protein 5UP5 inhibitor candidate modeled as structurally attached. There are a total of 10 different orientations of LOXL2 attached to mini-protein 5UP5. It can be seen that the attachment location varies significantly but the prime candidates to be effective over the catalytic location can be seen in diagrams 3, 7, and 9 whereas the other locations of attachment would probably not be effective in scaffolding the catalytic location of LOXL2 and Collagen-1. Structural images were generated using Pymol (134).

In figure 7, it denotes the 10 different attachment sequence locations of mini-protein 5UP5 to LOXL2 as simulated in Pymol. Overall 5UP5 had an average surface area binding of 61% which makes it one of the best candidates overall. As mentioned above, it is also important to try to have a scaffold that covers the enzymatic catalytic binding site where Collagen-1 and LOXL2 react. Location wise I can derive that model 3 which has a surface area binding of 67% and model 7 with a surface area binding of 73% could be effective LOXL2 inhibitors. Mini-proteins 5UP5 and 5UYO have shown to have the best geometric fit with LOXL2, which makes them both good candidates for further testing. Compared to the results from other mini-proteins, 5UYO has a much larger range of SASA percentage across its models, which may mean that it might not be as good of a candidate as 5UP5 is for scaffolding due to its more inconsistent results. However, 5UP5 also has the highest

percentage of geometric fit in binding schemes 3 and 7, which could still make it worth further testing alongside 5UYO with good geometric fit in binding schemes 3, 6, and 7.

CAF Targeted Gold Nanoparticle Delivery Motif Design Delivery of the mini-protein inhibitor to pre-catalyzed LOX in the ECM is key for the successful efficacy of this treatment motif. It is paramount that the selected mini-protein LOXL2 inhibitor be released in the ECM, ideally near CAFs where LOXL2 and collagen-1 are being released and undertaking cross-linking. This location can be saturated with LOXL2 inhibitors, and in theory, it will reduce or prevent collagen-1 cross-linking. For this reason, targeting the delivery of a gold nanoparticle 20 NM in length coated with an initial bilayer of LOXL2 mini-protein inhibitor, then secondarily coated with a FAP antibody Fv fragment bilayer and Notch/CL to target alpha-smooth muscle actin that is found to be abundant around CAFs. Then on the caps have an albumin deposition, which should allow the nanoparticle enough time to reach its target before being naturally eliminated in the body. Below is a primary design proposed to be used as a CAF targeted gold nanoparticle mini-protein inhibitor delivery motif design.

Figure 8: Proposed gold nanomaterial delivery design

DISCUSSION

This research was conducted to develop further an understanding of the potential use of mini-proteins as inhibitors for LOXL2 to slow down cancer metastasis. I tested eight mini-proteins: 5UOI, 5UP1, 6VGB, 5UP5, 5UYO, 6W90, 6VGA, and 6VG7 for their geometrical compatibility through SASA to determine which protein would be the best potential candidate to move forward for the next engineering steps. Out of the eight, 5UP5 and 5UYO had the highest SASA percentage at 61%, making these mini-proteins good candidates for being further tested as scaffolds for engineering a LOXL2 inhibitor. All of my findings are in silico, so the results of this research are only the first steps of the long engineering pipeline for making a real therapeutic, and many further steps would need to be taken before a well- developed LOXL2 inhibitor can be FDA approved. The crystalline structure of LOXL2 is a relatively new one, and although it enables us to create de novo binder designs, there is still very little known about the mechanisms of LOXL2, so it would be difficult to fully understand how a successfully designed inhibitor interacts with the protein. However, this will likely become known in the coming years, which could further guide the LOXL2 inhibitor design. As my results were based on computational design using docking tools such as ClusPro and PyMOL, docking collagen-1 to LOXL2 deemed to be difficult and unaccommodated for the tools used, as collagen is a semi-infinite fiber that docking tools do not recognize as such. This caused a discrepancy in my SASA results for docking collagen-1 and LOXL2. The results were much lower than anticipated for a receptor and a ligand that commonly bind together in nature and should be geometrically compatible as a result. From this research, the next steps will include measuring binding affinity between an inhibitor and the protein through binding KD , in vitro testing through AlphaLISA assays, and later in vivo testing through animal trials leading to clinical trials. Many factors contribute to the result of a primary tumor site becoming malignant, but by inhibiting excess LOXL2, the goal is to slow down invasion and metastatic spread of cancer, allowing current methods of cancer treatment to be more effective when cancer is in either a pre-metastatic or metastatic state.

CONCLUSION To make LOXL2 inhibition a useful treatment candidate, it needs to be a highly targeted approach of only inhibiting LOXL2 in tumor microenvironments near CAFs where the LOXL2 is secreted and can be inhibited near the source before any significant cross-linking can occur. If the near proximity of CAFs can be targeted this should prove to be both the most effective and the least toxic and most effective method possible to inhibit LOXL2 from cross-linking collagen-1 in the ECM and disrupting invasion and metastasis. It should further be noted that it has been demonstrated that inhibition of LOXL2 in several studies has been shown to be effective in decreasing invasion and metastasis (130). With this in mind, by combining one or both of the mini-proteins we analyzed - 5UP5 and 5UYO - along with the gold nanoparticle delivery motif proposed, this treatment scheme could prove to be a highly effective inhibitor of LOXL2 in the tumor microenvironment, preventing restructuring of the ECM that promotes further restructuring, invasion and metastasis. An added benefit and additional goal is that this treatment motif advocates an efficient strategy not only limiting spread of malignant cancer cells, but also “softening” the ECM stroma around the cancer tumor enabling more precise and effective penetration for conventional chemotherapy and radiotherapy increasing the overall efficiency of existing treatments that are normally not as effective in later stages of cancer progression.

RECOMMENDATIONS

We recommend that next steps first include further testing mini-proteins 5UP5 and 5UY0 with in vitro testing to determine effectiveness in preventing collagen-1 cross-linking. This can be accomplished by first testing the combination of LOXL2 with collagen-1 and analyzing the level of successful cross-linking. The second test would be to take each of the proposed mini-proteins, introduce them to the same LOXL2 formulation with the idea of inhibiting the LOXL2 formulation, then introduce collagen-1 and analyze the resultant collagen cross- linking if any. From this experiment, it should be determinable whether 5UP5 or 5UY0 were effective at inhibiting LOXL2 from collagen cross-linking, and which, if any of the two, are the best. If shown to have efficacy toward inhibition of LOXL2 to collagen-1 cross-linking it would then be beneficial to introduce the proposed gold nanoparticle targeted CAF delivery motif combined with the most effective mini-protein LOXL2 inhibitor and test and analyze in vivo cross-linking and CAF quiescence effectiveness.

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