1 Nikokasa

Elec 2607 Assignment 1 Skeletal System

Introduction


The contraction of cardiomyocytes has been studied extensively because it is crucial to proper heart function. Cross-bridges between actin and myosin lead to force generation and subsequently contraction. One important regulatory protein in the thin filament complex is troponin (Tn). Tn consists of three subunits: troponin C (TnC), troponin I (TnI), and troponin T (TnT).(1) Upon binding of the signaling ion, Ca2+, the N-terminal regulatory domain of TnC undergoes a structural reorganization that initiates myofilament contraction.(2) Calcium binding to TnC exposes a hydrophobic patch on TnC’s surface that promotes its association with the switch region of Troponin I (TnI). This in turn disturbs the interaction of the TnI inhibitory region with tropomyosin and actin, hence relieving TnI’s inhibition of contractile activity.(2, 3) Therefore, Ca2+-binding to the 89 residue N-terminal regulatory domain of TnC presents a crucial step in a chain of events leading to a contractile response. Elucidating its structure and dynamics is central to studies of the role of calcium in myofilament contraction.

A plethora of structural studies have been carried out to elucidate the structure and function of the TnC regulatory domain of which nuclear magnetic resonance (NMR) has been particularly popular.(4-7) Three states have been identified that are considered to be important intermediates in the myofilament contraction process: the free (apo) state, the Ca2+-bound state, and the Ca2+-TnI-switch-peptide-bound state. Additionally, the structures of the TnC regulatory domain in complex with small molecules(8) and mutants of TnC have been elucidated. The regulatory domain, a highly α-helical molecule that constitutes the N-terminal half of the troponin C protein, consists of five α-helices (N, A–D). Helices A through D comprise two EF-hand helix–loop–helix motifs. The EF-hands, which are known to be metal-binding sites, are labeled sites I and II.(9) In contrast with the C-terminal domain of TnC, which contains two high-affinity Ca2+-binding sites, the sites in the regulatory domain are of considerably lower affinity. Interestingly, in cardiac TnC, site I is completely defunct for calcium binding that is due to several amino acid substitutions with respect to site I in skeletal TnC.(10) Site II, the low-affinity, Ca2+-specific Ca2+-binding site, is generally considered to be the only site directly involved in calcium regulation of cardiac muscle contraction.(11) Ca2+-binding to site II of cardiac TnC does not induce an opening transition akin to skeletal TnC(12) but leaves the structure more or less unperturbed in the closed conformation.(4, 13) It is believed that the TnI switch peptide has to be present to stabilize the open conformation of the Ca2+-bound regulatory domain of cardiac TnC,(5, 14) suggesting that the open conformation may only be a transient state that is sampled by TnC after Ca2+-binding.

In addition to the structural properties of TnC, its calcium exchange kinetics have been the focus of much experimental investigation.(15) The calcium association rate to site II of isolated cardiac troponin C has been measured using stopped-flow techniques to be very high.(16-18) Similarly, stopped-flow techniques(16, 19)and NMR spectroscopy(20) were able to determine Ca2+-dissociation rates from site II much faster than the time scale of muscle relaxation.

Although a wide variety of experimental techniques have shed light on the structure and function of TnC, atomistic level simulations of TnC structure and dynamics in various states of calcium and TnI switch peptide association are necessary for understanding the binding processes involved. As elucidation of the dynamics of the TnC regulatory domain in response to calcium binding is integral for estimating Ca2+ affinity, this work may also provide a platform for computationally calculating association and dissociation rates. Additionally, relaxed complex scheme type approaches(21) could be used to generate actionable pharmaceutical leads by docking drug-like small molecules into structurally representative snapshots from molecular dynamics trajectories. In general, therapeutic strategies aim to improve calcium affinity in compromised tissue by changing the thermodynamics and kinetics of Ca2+ binding and TnI association. In this study, we use conventional and accelerated molecular dynamics (MD) to elucidate the dynamics of TnC with and without Ca2+ bound as well as with the TnI switch peptide bound. Comparison of molecular dynamics trajectories with experimentally obtained order parameters(22) is used as an assessment of how well the conformational landscape conducive to Ca2+ binding is sampled. We examine TnC for its ability of binding Ca2+ and furthermore determine the molecular contributions to Ca2+ binding kinetics. The results of these studies can provide a framework for understanding impaired Ca2+ handling in TnC mutants and knowledge gained from this study will guide the improvement of inotropic pharmaceuticals that target TnC.

Material and Methods


System Preparation

Three different systems of human cardiac troponin C were prepared for simulations. The apo system was modeled based on model 13 from pdb entry 1SPY (89 residues(4)). The Ca2+-bound system was modeled based on model 14 from pdb entry 1AP4 (89 residues, Ca2+-ion4). The Ca2+-TnI-bound system was modeled based on model 18 from pdb entry 1MXL (106 residues, Ca2+-ion5). All systems were neutralized adding Na+ counterions (1AP4: 13 Na+, 1SPY: 15 Na+, 1MXL: 11 Na+) and solvated using a TIP3P water box. The fully solvated systems contained 24 316 (1AP4), 26 756 (1SPY), and 25 994 (1MXL) atoms, respectively. Minimization using SANDER(23) was performed in two stages: 1000 steps of minimization of solvent and ions (the protein is restrained using a force constant of 500 kcal/mol/Å2), followed by a 2500 step minimization of the entire system. A short initial 20 ps MD simulation with weak restraints (10 kcal/mol/Å2) on the protein residues was used to heat up the system to a temperature of 300 K.

Conventional MD and Accelerated MD Simulations

Both cMD and aMD simulations were performed under the NPT ensemble at 300 K for all three TnC systems using AMBER(23) and the ff99SB force field.(24) Periodic boundary conditions were used, along with a nonbonded interaction cutoff of 10 Å. Bonds involving hydrogen atoms were constrained using the SHAKE algorithm,(25) allowing for a time step of 2 fs. For each system, 100 ns MD trajectories were generated (for the Ca2+-bound system MD a trajectory of even 150 ns) as well as 50 ns aMD trajectories at four different acceleration levels. Acceleration parameters were determined based on average potential and dihedral energies of the equilibrated MD simulations. Dual boost aMD (both the dihedral energy and the total potential energy are boosted) was used. The acceleration level is defined in terms of Eb and α, where Eb is the threshold boost energy and α is a tuning parameter that determines the shape of the accelerated potential.(26) On the basis of a comparative analysis of previous successful aMD studies(27-29) on a variety of systems of different sizes, the optimal boost energy for the torsional aMD is usually found to be the average dihedral angle energy plus 3.5 times the number of residues in the solute, and α should be ∼20% of Eb. Similarly the acceleration parameters for the acceleration of the total potential energy are dependent on the number of atoms in the entire simulation cell.(30, 31) For the aMD simulations, a new in-house version of PMEaMD was used.

Clustering

Frames every 6 ps were extracted from the MD trajectories. The frames were aligned using all Cα atoms in the protein and subsequently clustered by rmsd using GROMOS++ conformational clustering.(32) An rmsd cutoff of 1.6 Å (1AP4), 1.7 Å (1SPY) and 1.8 Å (1MXL) was chosen, respectively. These cutoffs resulted in 7 (1AP4), 9 (1SPY), and 8 (1MXL) clusters that represented at least 90% of the respective trajectories. The central members of each of these clusters were chosen to represent the protein conformations within the cluster and thereby the conformations sampled by the trajectory.

Principal Component Analysis

The principal component analysis (PCA) was performed using the bio3D package in R.(33) A blast profile of the 1AP4 sequence revealed 1056 hits. Out of the 35 most similar hits, 29 hits were chosen (excluding structures with mutations and the recently crystallized cadmium coordinating structure(34)), and their experimentally determined structures were obtained from the protein data bank. The 29 structures underwent iterative rounds of structural superposition to determine the invariant core of the protein, a region that exhibits the least structural variance between the protein structures. This core consists of residues 17–28 and 72–79. Subsequently, the experimental structures were superimposed onto this core, and a PCA was employed.(35, 36) In this process, a covariance matrix from the coordinates of the superimposed structures is diagonalized. The eigenvectors of this matrix represent the principal components of the system (parts of the structure within which there is the most variety among the set of superimposed experimental structures), whereas the eigenvalues are a measure of the variance within the distribution along the respective eigenvectors. All experimental structures have been projected into the space spanned by principal components one and two (along which there is the most variance among the structures). The principal component space generated based on the similar experimental structures served as basis for projection of the molecular dynamics trajectories. For the analysis of interhelical angles, interhelical angles were calculated using interhlx (K. Yap, University of Toronto).

Simulation of NMR Observables

On the basis of the MD trajectory of the Ca2+-bound system (1AP4), chemical shifts for the amide 1H and the amide 15N were calculated using the SHIFTX software.(37) Protein structures were extracted from the trajectory every 20 ps. For each of these structures chemical shifts were calculated and averaged. Backbone N–H order parameters were calculated from the 100 ns molecular dynamics simulation of the apo system (1SPY) using the isotropic reorientational eigenmode dynamics (iRED) approach.(38) Order parameters were calculated by averaging 0.5 ns trajectory windows to ensure that the calculated S2 parameters do not contain any motions whose time scale exceeds the overall tumbling correlation time of the protein.(39) The ptraj program was used to generate a list of eigenvalues and eigenvectors from all of the N–H backbone vectors with the ired method. On the basis of these lists, the order parameters are calculated using the mat2s2.py script.

Brownian Dynamics Simulations

BrownDye was used to estimate calcium association rates.(40) PQR files for representative protein structures determined by a cluster analysis were generated using pdb2pqr.(41) The calcium pqr file was generated using a charge of +2 and an ionic radius of 1.14 Å. APBS(42) was used to generate the electrostatic fields for the protein and the calcium ion in openDX format. Bd_top was used to generate all necessary input files for the BrownDye runs. A phantom atom of zero charge and negative radius (−1.14 Å) was introduced after the first execution of bd_top. The phantom atom was placed at the position of the calcium ion from the trajectory frame. It has no influence on the association rate constant calculation and serves solely to be able to define a reaction criterion that is spherically symmetric around the expected binding position of the calcium. The reaction criterion was chosen to be 1.2 Å within the calcium binding site. We performed 500 000 single trajectory simulations on 8 parallel processors using nam_simulation. The reaction rate constants were calculated using compute_rate_constant from the BrownDye package. A weighted average of the rate constants of each of the representative cluster centers yielded an estimate of the overall rate constant for the system.

Results & Discussion


Sampling by Conventional MD versus Accelerated MD

PCA characterizes collective, high-amplitude structural variations based on a set of homologous protein structures. The predominant modes provide a basis for analyzing large-scale conformational changes anticipated in MD simulations. In this study, NMR structures of apo-TnC, Ca2+-bound TnC, Ca2+/TnI-bound TnC, and TnC in complex with compounds such as bepridil, W7, and dfbp were used as inputs for PCA. The first two principal components, PC1 and PC2, are illustrated in Figure 1and account for 50.9 and 13.5%, respectively, of the variance associated with known TnC structures. As the two components together account for 64.4% of the variance, it was considered to be appropriate to analyze the simulations just in terms of these components. PC3 accounts for another 10.4% of the variance but was not used for analysis because of its relatively low contribution. The quadrants along PC1 and PC2 describe apo and Ca2+-bound TnC structures (lower right, e.g., 1SPY, 1AP4) and Ca2+-TnI-bound TnC structures (left side, e.g., 1MXL, 2L1R).

Table of Contents

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PROSPECTUS SUMMARY

This summary highlights information contained elsewhere in this prospectus and does not contain all of the information that you should consider in making your investment decision. Before investing in our common stock, you should carefully read this entire prospectus, including our consolidated financial statements and the related notes included elsewhere in this prospectus. You should also consider, among other things, the matters described under “Risk Factors” and “Management’s Discussion and Analysis of Financial Condition and Results of Operations,” in each case appearing elsewhere in this prospectus. Unless otherwise stated, all references to “us,” “our,” “aTyr,” “we,” the “Company” and similar designations refer to aTyr Pharma, Inc. and its subsidiary, Pangu BioPharma Limited.

Overview

We engage in the discovery and clinical development of innovative medicines for patients suffering from severe, rare diseases using our knowledge of Physiocrine biology, a newly discovered set of physiological modulators. We have discovered approximately 300 Physiocrines (physio for life and crine for specific activity), a class of naturally occurring proteins that we believe promote homeostasis, a fundamental process of restoring stressed or diseased tissue to a healthier state. Physiocrines are extracellular signaling regions of tRNA synthetases, an ancient family of enzymes that catalyze a key step in protein synthesis. We believe that Physiocrines have evolved over time to modulate important cellular pathways by interacting with various types of cells, including immune and stem cells. Approximately 100 of these proteins interact with the immune system, which we believe presents a significant therapeutic opportunity to restore affected tissues to a healthier state through natural immuno-modulation mechanisms. We successfully completed a Phase 1 clinical trial of Resolaris, our first development candidate from our discovery engine, and are currently conducting a multi-national exploratory Phase 1b/2 clinical trial of Resolaris in adult patients with facioscapulohumeral muscular dystrophy, or FSHD, a severe, rare genetic myopathy with an immune component, for which there are currently no approved treatments. By leveraging our discovery engine and our knowledge of rare diseases, we aim to build a proprietary pipeline of novel product candidates with the potential to treat severe, rare diseases characterized by immune dysregulation. We plan to independently commercialize our Physiocrine-based therapeutics.

Our scientists were the first to identify the Resokine pathway (reso for restoring skeletal muscle health and kine for activity related to cytokines), an extracellular pathway in human skeletal muscle tissue associated with activities arising from various Physiocrine regions of the histidine aminoacyl tRNA synthetase, or HARS. We believe the Resokine pathway may play an important role in muscle and lung health. Certain patients with antisynthetase syndrome, a rare auto-immune disease, have antibodies to HARS, which are known as Jo-1 antibodies. These Jo-1 antibody patients often develop two significant clinical manifestations, skeletal inflammatory myopathy and interstitial lung disease, or ILD. We believe that the binding of Jo-1 antibodies, particularly to the immuno-modulatory domain of HARS, or iMod domain, blocks HARS immuno-modulatory functions and results in the muscle and lung disease in these Jo-1 antibody patients.

We are harnessing the Resokine pathway and its association with homeostasis in skeletal muscle to develop Resolaris as a first-in-class therapeutic for patients with rare myopathies with an immune component, or RMICs, for which there are limited or no approved treatments. A myopathy is a disease of skeletal muscle tissue, characterized by muscle fiber deterioration, muscle weakness and often an immune response in the affected muscle tissue. In contrast to most current immunology drugs, which are engineered antagonists of immunological pathways, Resolaris is derived from a naturally occurring protein, HARS, which we believe has the potential to reset the immune system in diseased tissue to a more normal state while maintaining the immune system’s activity against exogenous, pathogen-based insults. We observed that stimulation of the Resokine pathway through the introduction of Resolaris and its derivatives in rodent models of both severe inflammation and myopathy led to immuno-modulatory effects. We have shown that stimulation of the Resokine pathway by Resolaris alters immune responses and the expression or release of immune-related proteins from cells in response to inflammation. HARS, which

 

contains the immuno-modulatory domain, is also released from human skeletal muscle. In addition to its immuno-modulatory properties, we believe the Resokine pathway may act on other physiological processes, including processes associated with stem cells, fibrosis and endothelial cells. Our initial therapeutic efforts target severe, rare disease indications in which patients suffer from the immune-related consequences of their genetic disease. We have identified over 20 distinct, molecularly definable RMIC indications, including FSHD and limb-girdle muscular dystrophies, or LGMD, in which we believe Resolaris has the potential to target the immune component of these genetic diseases.

We are also harnessing the Resokine pathway and its potential role in lung disease, specifically ILD, to develop Resolaris as a therapeutic for patients with rare pulmonary diseases with an immune component, or RPICs. ILD is associated with Jo-1 antibody patients and occurs in multiple other clinical settings. We are currently evaluating these other forms of ILD to identify the most appropriate RPIC indication for the initial clinical assessment of augmenting the Resokine pathway with Resolaris.

We have initiated a discovery program to explore varying exposures of the iMod domain of the Resokine pathway through protein engineering. The program seeks to develop a potential therapeutic that we refer to as iMod.Fc. We also believe our proprietary inventory of Physiocrines with their diverse functions have potential therapeutic application in a variety of diseases characterized by tissue dysfunction, including severe diseases of the lung, gut, skin, brain and liver. We intend to leverage our unique understanding of Physiocrines and their functions and our broad intellectual property portfolio, which we believe covers this entire class of potential protein therapeutics, to build a pipeline of product candidates that we expect to develop and commercialize independently for the treatment of various rare diseases.

Below are summaries of our product development pipeline and discovery engine process:

 

 

We were founded in 2005 by Paul Schimmel, Ph.D. and Xiang-Lei Yang, Ph.D., two leading aminoacyl tRNA synthetase scientists at The Scripps Research Institute in San Diego, California. Our Executive Chairman and Chief Executive Officer, John D. Mendlein, Ph.D., was formerly the Chief Executive Officer of Adnexus Therapeutics, Inc. (acquired by Bristol-Myers Squibb Company) and Affinium Pharmaceuticals, Ltd. (acquired by Debiopharm Group), and held various roles at Aurora Biosciences Corporation (acquired by Vertex Pharmaceuticals, Incorporated). We have assembled an executive team with broad experience in the discovery, development and commercialization of innovative therapeutics, including transformative therapies for rare genetic diseases, such as Kalydeco, marketed by Vertex Pharmaceuticals Incorporated for the treatment of cystic fibrosis. We are advised by a Therapeutic Advisory Board and a Scientific Advisory Board, both comprised of leaders in the field of biology for medical applications, including our special advisor in immunology, Bruce Beutler, M.D., recipient of the 2011 Nobel Prize in Physiology or Medicine for his work in immunology. Our key investors include entities affiliated with Alta Partners; Cardinal Partners; Domain Associates; Fidelity Management & Research Company; Polaris Partners and Sofinnova Ventures.

Our Physiocrine Advantage: Targeting the Immune System in Genetic Diseases

We believe the immune system is an important component of the pathophysiology of many rare genetic diseases. It is our belief that the immune system acts differently in the presence of some genetic mutations that alter protein levels, structure or function compared to normal tissue. This immune response contributes to a pathophysiologic state in the diseased tissue. By modulating various components of the immune system, Physiocrines can potentially alter this pathophysiological immune activity in the diseased tissue by promoting homeostasis and restoring immune balance in the diseased tissue. Using the immune component as a target or intervention point in the treatment of genetic diseases has precedent as an approach to developing a protein therapeutic. Examples include Soliris, for acquired paroxysmal nocturnal hemoglobinuria (PNH), and Cinryze, for hereditary angioedema (HAE).

Resolaris, Our First Clinical Product Candidate: a Pipeline within a Product Opportunity

Resolaris in FSHD, a Rare Myopathy with an Immune Component (RMIC)

We developed Resolaris based on our discovery of the Resokine pathway in skeletal muscle tissue, an extracellular pathway in human skeletal muscle tissue associated with activities arising from various Physiocrine regions of the human histidine aminoacyl tRNA synthetase. We believe, based on preclinical data and observations from Jo-1 antibody patients, that the Resokine pathway is involved in promoting skeletal muscle health and homeostasis. We believe it does so, in part, by acting as an immunomodulator in skeletal muscle.

Our first clinical development target for Resolaris is FSHD, a rare genetic myopathy in which immune cells invade diseased skeletal muscle and for which there are no approved treatments. The primary clinical phenotype of FSHD is debilitating skeletal muscle deterioration and weakness. The symptoms of FSHD develop in an asymmetrical “muscle by muscle” fashion. This is in contrast to other genetic myopathies, such as Duchenne muscular dystrophy, that usually affect groups of muscles concurrently and symmetrically. In addition to debilitating muscle weakness, FSHD patients often experience severe fatigue, muscle deterioration and pain. The disease is typically diagnosed by the presence of a characteristic pattern of muscle weakness and other clinical symptoms, as well as through genetic testing. While estimates of FSHD prevalence vary, studies exploring the topic have identified average prevalence rates of approximately one in 17,000. Applying this rate to the U.S. population, based on recent census data, yields a domestic FSHD population of approximately 19,000.

We successfully completed a single ascending dose Phase 1 clinical trial in healthy subjects of Resolaris in the first quarter of 2014. Resolaris was found to be well tolerated in all dose cohorts and there were no serious adverse events. We are currently conducting a multi-national exploratory Phase 1b/2 clinical trial of Resolaris in

 

adult patients with FSHD in the European Union. This randomized, double-blind, placebo-controlled trial is designed to evaluate the safety, tolerability, pharmacokinetics and immunogenicity of multiple intravenous doses of Resolaris in adults with FSHD. We also intend to explore pharmacodynamic changes in immune activity and responses in skeletal muscle. Resolaris is being studied in three dose escalation cohorts (0.3 mg/kg, 1.0 mg/kg and 3.0 mg/kg). In the fourth quarter of 2014, we completed multiple dosing of the patients in the first dose cohort. We are currently dosing patients in the second cohort. Subject to our interactions with regulatory authorities and patient enrollment in accordance with our clinical development plans, we expect to report initial results from this clinical trial in the fourth quarter of 2015 or early 2016. In parallel with conducting our initial clinical trial in adults with FSHD, we are finalizing our plans to evaluate Resolaris in a multi-center, international trial of patients with early onset FSHD, which we define as patients with onset of disease before the age of 18. Subject to our interactions with regulatory authorities, we expect to initiate this clinical trial in the third quarter of 2015.

Resolaris in Other RMIC Indications

In addition to FSHD, we plan to address other severe, genetic diseases in which immune cells invade diseased muscle. We are evaluating various forms of limb-girdle muscular dystrophy, or LGMD, a broad class of indications of over 20 rare genetically defined myopathies. These diseases are linked by the common distribution of their muscle weakness (e.g., predominantly in the proximal limb muscles and the pelvic and shoulder girdle muscles). We intend to select genetic forms of LGMD that we believe will be most amenable to treatment with Resolaris, such as those with the characteristics of the associated immuno-pathology in skeletal muscle. We plan to commence clinical trials of Resolaris in at least one LGMD indication in adult patients in the fourth quarter of 2015.

Resolaris Non-Muscle Indication Set: Rare Pulmonary Diseases with an Immune Component (RPICs)

The Resokine pathway may play an important role in lung health. ILD develops in approximately 85% of anti-synthetase syndrome patients with Jo-1 antibodies to Resokine. In addition to its association with Jo-1 antibody patients, ILD occurs in multiple other clinical settings. We are currently evaluating these forms of ILD to identify the most appropriate RPIC indication for the initial clinical assessment of Resolaris. Among these forms of ILD, we have identified several that can result in severe and progressive lung disease and share immuno-pathophysiology features that overlap with our demonstrated Resolaris activities. Examples include idiopathic non-specific interstitial pneumonias, idiopathic pulmonary fibrosis, lymphocytic interstitial pneumonia, bleomycin (the chemotherapeutic agent)-induced pulmonary fibrosis, and ILD in the setting of systemic sclerosis, or scleroderma, and sarcoidosis.

To test that augmenting the Resokine pathway has therapeutic potential in ILD, we have recently generated data in a mouse model of lung inflammation and pulmonary fibrosis induced by bleomycin. The mouse equivalent of Resolaris has shown promising therapeutic activity in this model which has been used previously in the development of therapeutics for different forms of ILD, including the drug pirfenidone or Esbriet, which was approved by the FDA in October 2014 for the treatment of idiopathic pulmonary fibrosis. We noted that Resolaris administration attenuated the radiographic and histological manifestations of pathophysiology in this model when it was dosed therapeutically. These mouse Resolaris pharmacology data provide pre-clinical evidence supporting the therapeutic potential of Resolaris for the treatment of ILD.

We are currently evaluating the most appropriate RPIC indication for the initial clinical evaluation of augmenting the Resokine pathway in lung via Resolaris. The data obtained in this initial ILD trial will inform further development of therapeutics leveraging the Resokine pathway in RPICs.

 

An Emerging Pipeline of Product Opportunities

Our Preclinical Immuno-Modulatory Domain Program from the Resokine Pathway: iMod.Fc

We have conducted a series of experiments to understand how various product form modifications enhance exposure and activity of the iMod domain of Resokine. Fc fusion proteins have been successfully commercialized previously by others to enhance exposure while enabling biological activity. We explored this approach by fusing the immunoglobin Fc with one iMod domain, which can form a dimer.

Our Fc fusion experiments have begun to delineate how to enhance the exposure of the iMod domain of Resokine while maintaining activity and provide insights into this domain harboring immuno-modulatory activity. Initial experiments have indicated that Fc fusion proteins can increase exposure and maintain iMod domain activity. We have generated encouraging results for one iMod.Fc in a mouse model of lung inflammation and fibrosis.

Our Discovery Engine for Therapeutic Applications of Physiocrines: Lung and Liver Focused

Our discovery efforts are based on our scientific investigation of Physiocrine pathways. Through a combination of deep sequencing and bioinformatics panning, augmented by proteomic analysis, we identified over 300 naturally occurring Physiocrines. We expressed and purified over 200 of these Physiocrines and evaluated these purified Physiocrines in numerous cell-based assays to determine their activity in important human physiological pathways. In July 2014, a publication in Science described a portion of the results from our research, along with our collaborators at Scripps La Jolla, Scripps Florida, Stanford University and the Hong Kong University of Science and Technology.

Our scientists have conducted experiments that demonstrated that the blockade of Physiocrine pathways in rodents resulted in an in vivo phenotype characterized by immune cell infiltration or fibrotic disease in the lung or the liver. These data support the concept that Physiocrines may have the potential to inhibit, limit, or otherwise regulate immune cell activity in both the lung and the liver, as well as the subsequent development of fibrosis in these tissues. Accordingly, we are continuing to investigate certain Physiocrines for potential therapeutic applications in both lung and liver indications.

Our Strategy

We aim to capitalize on Physiocrine biology, a new and important area of human health, to develop first-in-class medicines to treat patients with severe diseases characterized by an immune component. Key elements of our strategy include the following:

 

  Leverage our leadership position in Physiocrine biology to develop and commercialize novel, first-in-class medicines for patients affected by severe, rare diseases with significant unmet need. We believe our initial focus on severe, rare diseases will allow us to more effectively deploy investor capital for the independent development and commercialization of medicines for the benefit of patients and our stakeholders.

 

  Rapidly and prudently pursue the development and commercialization of Resolaris to treat patients across multiple severe, rare disease indications. We are currently evaluating Resolaris in a Phase 1b/2 clinical trial in adult patients with FSHD and expect to report initial results from this clinical trial in the fourth quarter of 2015 or early 2016. In addition, we plan to initiate clinical trials of Resolaris in early onset FSHD and other RMIC indications, including LGMD, as well as other rare diseases with an immune component, such as RPIC indications.

 

  Leverage our discovery engine to build a pipeline of first-in-class Physiocrine medicines to address severe conditions characterized by immune pathway dysfunction or fibrosis. We plan to leverage our discovery engine to identify other Physiocrine pathways of interest and select additional potential product candidates for preclinical and clinical investigation in a variety of disease settings on a tissue-by-tissue basis, which may include severe, currently inadequately treated diseases of the lung and liver.

 

  Retain exclusive worldwide commercial rights to our product candidates to pursue autonomous commercialization. We intend to build a pipeline of product candidates that we can commercialize independently through a relatively small, dedicated commercial organization focused on patient needs and directed at a limited number of physicians who specialize in the treatment of our target patient populations.

 

  Expand our knowledge and intellectual property position in Physiocrine biology by emphasizing continuous scientific and business improvements. We intend to aggressively pursue new scientific and therapeutic insights into the potential therapeutic applications of Physiocrines, and to broaden our patent portfolio across this class of novel protein therapeutics and their antibody antagonists.

 

  Build a world class organization oriented to patients and focused on rigorous scientific, clinical and industrial advancements. We have assembled a world class team with industry-recognized expertise in biology, medicine and the commercialization of innovative and important therapeutics. We intend to continue to build on our leadership position in Physiocrine and immunology-based therapeutics and grow an organization and culture dedicated to the development and commercialization of medicines with the potential to positively transform the lives of patients with severe, rare diseases.

Risks Associated with Our Business

Our ability to implement our business strategy is subject to numerous risks, as more fully described in the section entitled “Risk Factors” immediately following this prospectus summary. These risks include, among others:

 

  Resolaris, and any other product candidates that we may develop, represent novel therapeutic approaches, which may cause significant delays or may not result in any commercially viable drugs.

 

  We are highly dependent on the success of Resolaris, which is still in early clinical development. If we are unable to successfully complete or otherwise advance clinical development, obtain regulatory or marketing approval for, or successfully manufacture or commercialize, Resolaris, or experience significant delays in doing so, our business will be materially harmed.

 

  Data generated in our preclinical studies and patient sample data relating to the Resokine pathway may not be predictive or useful for determining the immuno-modulatory activity or therapeutic effects, if any, of Resolaris in patients, and success in early-stage clinical trials may not be predictive of success in later-stage clinical trials.

 

  We have incurred significant losses since our inception and anticipate that we will continue to incur significant losses for the foreseeable future. We have never generated any revenue from product sales and may never be profitable.

 

  We will need substantial additional funding. If we are unable to raise capital when needed, we would be forced to delay, reduce or eliminate product development programs or commercialization efforts.

 

  We have not studied Resolaris or any of our other product candidates in any human clinical trials designed to show efficacy to date.

 

  We are developing novel product candidates for the treatment of diseases in which there is little clinical drug development experience and, in some cases, are using new endpoints or methodologies. The regulatory pathways for approval are not well defined, and as a result there is greater risk that the outcome of our clinical trials will not be favorable.

 

  We rely, and expect to continue to rely, on third parties to conduct some or all aspects of our product manufacturing, protocol development, research and preclinical and clinical testing, and these third parties may not perform satisfactorily.

 

  If we are unable to obtain and maintain patent, trade secret or other intellectual property protection for our medicines and technology, or if the scope of the patent protection obtained is not sufficiently broad, our competitors could develop and commercialize medicines and technology similar or identical to ours, and our ability to successfully commercialize our medicines and technology may be adversely affected.

 

  If we are not able to obtain, or if there are delays in obtaining, required regulatory approvals, we will not be able to commercialize, or will be delayed in commercializing, our product candidates, and our ability to generate revenue will be materially impaired.

Implications of Being an Emerging Growth Company

We qualify as an “emerging growth company” as defined in the Jumpstart Our Business Startups Act of 2012, as amended, or the JOBS Act. As an emerging growth company, we may take advantage of specified reduced disclosure and other requirements that are otherwise applicable generally to public companies. These provisions include:

 

  only two years of audited financial statements in addition to any required unaudited interim financial statements with correspondingly reduced “Management’s Discussion and Analysis of Financial Condition and Results of Operations” disclosure;

 

  reduced disclosure about our executive compensation arrangements;

 

  no non-binding advisory votes on executive compensation or golden parachute arrangements; and

 

  exemption from the auditor attestation requirement in the assessment of our internal control over financial reporting.

We may take advantage of these exemptions for up to five years or such earlier time that we are no longer an emerging growth company. We would cease to be an emerging growth company on the date that is the earliest of (i) the last day of the fiscal year in which we have total annual gross revenues of $1 billion or more; (ii) the last day of our fiscal year following the fifth anniversary of the date of the completion of this offering; (iii) the date on which we have issued more than $1 billion in nonconvertible debt during the previous three years; or (iv) the date on which we are deemed to be a large accelerated filer under the rules of the Securities and Exchange Commission, or SEC. We may choose to take advantage of some but not all of these exemptions. We have taken advantage of reduced reporting requirements in this prospectus. Accordingly, the information contained herein may be different from the information you receive from other public companies in which you hold stock. We have irrevocably elected to “opt out” of the exemption for the delayed adoption of certain accounting standards and, therefore, will be subject to the same new or revised accounting standards as other public companies that are not emerging growth companies.

Company and Other Information

We were incorporated under the laws of the State of Delaware in September 2005. Our principal executive office is located at 3545 John Hopkins Court, Suite #250, San Diego, California 92121, and our telephone number is (858) 731-8389. Our website address is www.atyrpharma.com. We do not incorporate the information on or accessible through our website into this prospectus, and you should not consider any information on, or that can be accessed through, our website as part of this prospectus.

 

THE OFFERING

 

Common stock offered by us

            shares.

 

Common stock to be outstanding immediately after this offering

            shares (             shares if the underwriters exercise their over-allotment option in full).

 

Underwriters’ option to purchase additional shares

We have granted a 30-day option to the underwriters to purchase up to an aggregate of             additional shares of common stock to cover over-allotments.

 

Use of proceeds

We intend to use the net proceeds from this offering to fund our clinical development of Resolaris, to advance our other research, discovery and development activities, and for working capital and general corporate purposes. For a more complete description of our intended use of the proceeds from this offering, see “Use of Proceeds.”

 

Risk factors

You should carefully read “Risk Factors” in this prospectus for a discussion of factors that you should consider before deciding to invest in our common stock.

 

Proposed NASDAQ Global Market symbol

“LIFE”

The number of shares of our common stock to be outstanding after this offering is based on 136,729,927 shares of our common stock outstanding as of December 31, 2014, which includes the conversion of all outstanding shares of redeemable convertible preferred stock, including the shares of our Series E redeemable convertible preferred stock issued in March 2015, into an aggregate of 129,492,356 shares of common stock immediately prior to the completion of this offering and excludes:

 

  12,047,225 shares of common stock issuable upon the exercise of stock options outstanding as of December 31, 2014 at a weighted average exercise price of $0.58 per share;

 

  206,581 shares of common stock issuable upon the exercise of warrants outstanding as of December 31, 2014 at a weighted average exercise price of $1.82 per share, which warrants prior to the completion of this offering are exercisable to purchase redeemable convertible preferred stock;

 

  the issuance of 953,228 shares of common stock to The Scripps Research Institute on March 31, 2015;

 

  2,388,777 shares of common stock issuable upon the exercise of stock options granted to employees, directors and consultants subsequent to December 31, 2014 at a weighted average exercise price of $1.15 per share;

 

  10,527,447 shares of common stock currently available for future issuance under our 2014 Stock Plan;

 

  751,314 shares of common stock issuable upon the conversion of 751,314 shares of Series D redeemable convertible preferred stock that may be issued under a convertible promissory note issued to an affiliate of our landlord, if the noteholder elects to convert the note in accordance with its terms; and
                  shares of common stock reserved for future issuance under our 2015 Stock Option and Incentive Plan, or the 2015 Plan, which will become effective immediately prior to the completion of this offering.

 

Unless otherwise indicated, all information in this prospectus reflects or assumes the following:

 

  the filing of our amended and restated certificate of incorporation and the adoption of our amended and restated bylaws, which will occur immediately prior to the completion of this offering;

 

  the issuance and sale of 68,166,894 shares of our Series E redeemable convertible preferred stock in March 2015 for aggregate gross proceeds of approximately $76.3 million;

 

  the conversion of all of our outstanding shares of redeemable convertible preferred stock, including the shares of our Series E redeemable convertible preferred stock issued in March 2015, into 129,492,356 shares of common stock upon the completion of this offering at a rate of one share of redeemable convertible preferred stock into one share of common stock, except for our Series E redeemable convertible preferred stock, for which the conversion rate is one share of Series E redeemable convertible preferred stock into approximately 0.8216 of a share of common stock;

 

  our repayment in cash, upon the completion of this offering, of approximately $2.5 million in principal and accrued interest as of December 31, 2014 under a convertible promissory note issued to an affiliate of our landlord, assuming the note holder does not elect, on or prior to the date of completion of this offering, to forgive all accrued interest under the note and convert the $2.0 million in principal under the note into 751,314 shares of our Series D redeemable convertible preferred stock, which would convert into 751,314 shares of common stock upon the completion of this offering; and

 

  no exercise by the underwriters of their option to purchase up to an additional             shares of common stock in this offering.

 

SUMMARY CONSOLIDATED FINANCIAL DATA

The following summary consolidated financial information should be read together with the information under the caption “Management’s Discussion and Analysis of Financial Condition and Results of Operations” and our consolidated financial statements and accompanying notes appearing elsewhere in this prospectus. The summary consolidated statement of operations data for the years ended December 31, 2013 and 2014 and the summary consolidated balance sheet data as of December 31, 2014 are derived from our audited consolidated financial statements appearing elsewhere in this prospectus. Our historical results are not necessarily indicative of results that may be expected in the future.

 

   Years Ended
December 31,
 
   2013  2014 
   (in thousands, except share
and per share data)
 

Statements of Operations Data:

   

Operating expenses:

   

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