Considerations for design, manufacture, and delivery for effective and safe T-cell engager therapies.

T-cell engager (TCE) molecules provide a targeted immunotherapy approach to treat hematologic malignancies and solid tumors. Since the approval of the CD19-targeted BiTE® (bispecific T-cell engager) molecule blinatumomab, multiple TCE molecules against different targets have been developed in several tumor types, with the approval of three additional TCE molecules in 2022. Some of the initial challenges, such as the need for continuous intravenous administration and low productivity, have been addressed in subsequent iterations of the platform by advancing half-life extended, Fc-based molecules. As clinical data from these molecules emerge, additional optimization of formats and manufacturability will be necessary. Ongoing efforts are focused on further improving TCE efficacy, safety, and convenience of administration.

Systematic Study of the Glutathione Reactivity of N-Phenylacrylamides: 2. Effects of Acrylamide Substitution.

A comprehensive understanding of structure-reactivity relationships is critical to the design and optimization of cysteine-targeted covalent inhibitors. Herein, we report glutathione (GSH) reaction rates for N-phenyl acrylamides with varied substitutions at the α- and ß-positions of the acrylamide moiety. We find that the GSH reaction rates can generally be understood in terms of the electron donating or withdrawing ability of the substituent. When installed at the ß-position, aminomethyl substituents with amine pKa's > 7 accelerate, while those with pKa's < 7 slow the rate of GSH addition at pH 7.4, relative to a hydrogen substituent. Although a computational model was able to only approximately capture experimental reactivity trends, our calculations do not support a frequently invoked mechanism of concerted amine/thiol proton transfer and C-S bond formation and instead suggest that protonated aminomethyl functions as an electron-withdrawing group to reduce the barrier for thiolate addition to the acrylamide.

The immunomodulatory drugs lenalidomide and pomalidomide enhance the potency of AMG 701 in multiple myeloma preclinical models.

We investigated here the novel immunomodulation and anti-multiple myeloma (MM) function of T cells engaged by the bispecific T-cell engager molecule AMG 701, and further examined the impact of AMG 701 in combination with immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide). AMG 701 potently induced T-cell-dependent cellular cytotoxicity (TDCC) against MM cells expressing B-cell maturation antigen, including autologous cells from patients with relapsed and refractory MM (RRMM) (half maximal effective concentration, <46.6 pM). Besides inducing T-cell proliferation and cytolytic activity, AMG 701 also promoted differentiation of patient T cells to central memory, effector memory, and stem cell-like memory (scm) phenotypes, more so in CD8 vs CD4 T subsets, resulting in increased CD8/CD4 ratios in 7-day ex vivo cocultures. IMiDs and AMG 701 synergistically induced TDCC against MM cell lines and autologous RRMM patient cells, even in the presence of immunosuppressive bone marrow stromal cells or osteoclasts. IMiDs further upregulated AMG 701-induced patient T-cell differentiation toward memory phenotypes, associated with increased CD8/CD4 ratios, increased Tscm, and decreased interleukin 10-positive T and T regulatory cells (CD25highFOXP3high), which may downregulate T effector cells. Importantly, the combination of AMG 701 with lenalidomide induced sustained inhibition of MM cell growth in SCID mice reconstituted with human T cells; tumor regrowth was eventually observed in cohorts treated with either agent alone (P < .001). These results strongly support AMG 701 clinical studies as monotherapy in patients with RRMM (NCT03287908) and the combination with IMiDs to improve patient outcomes in MM.

AMG 701 induces cytotoxicity of multiple myeloma cells and depletes plasma cells in cynomolgus monkeys.

Multiple myeloma (MM) is a hematologic malignancy that is characterized by the accumulation of abnormal plasma cells (PCs) in the bone marrow (BM). Patient outcome may be improved with BiTE (bispecific T-cell engager) molecules, which redirect T cells to lyse tumor cells. B-cell maturation antigen (BCMA) supports PC survival and is highly expressed on MM cells. A half-life extended anti-BCMA BiTE molecule (AMG 701) induced selective cytotoxicity against BCMA-expressing MM cells (average half-maximal effective concentration, 18.8 ± 14.8 pM), T-cell activation, and cytokine release in vitro. In a subcutaneous mouse xenograft model, at all doses tested, AMG 701 completely inhibited tumor formation (P < .001), as well as inhibited growth of established tumors (P ≤ .001) and extended survival in an orthotopic MM model (P ≤ .01). To evaluate AMG 701 bioactivity in cynomolgus monkeys, a PC surface phenotype and specific genes were defined to enable a quantitative digital droplet polymerase chain reaction assay (sensitivity, 0.1%). Dose-dependent pharmacokinetic and pharmacodynamic behavior was observed, with depletion of PC-specific genes reaching 93% in blood and 85% in BM. Combination with a programmed cell death protein 1 (PD-1)-blocking antibody significantly increased AMG 701 potency in vitro. A model of AMG 701 binding to BCMA and CD3 indicates that the distance between the T-cell and target cell membranes (ie, the immunological synapse) is similar to that of the major histocompatibility complex class I molecule binding to a T-cell receptor and suggests that the synapse would not be disrupted by the half-life extending Fc domain. These data support the clinical development of AMG 701.

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms.

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

Nonclinical Safety Assessment of AMG 553, an Investigational Chimeric Antigen Receptor T-Cell Therapy for the Treatment of Acute Myeloid Leukemia.

Feline McDonough Sarcoma-like tyrosine kinase 3 (FLT3), a tyrosine-protein kinase involved in hematopoiesis, is detectable on the cell surface of approximately 80% of leukemia isolates from adult patients with acute myeloid leukemia (AML). AMG 553 is an investigational chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of AML. FLT3 expression analysis and in vitro and in vivo studies were leveraged to evaluate the nonclinical safety of AMG 553. Cynomolgus monkeys administered autologous anti-FLT3 CAR T cells demonstrated no evidence of CAR T-cell-mediated toxicity, expansion, or persistence, likely due to restricted cell surface FLT3 protein expression in healthy animals. This highlights the limited value of such in vivo studies for safety assessment of the CAR T-cell modality when directed against a target with restricted expression. To complement these studies and directly evaluate the potential toxicities of eliciting T-cell-mediated cytotoxicity against cells with surface expression of FLT3 protein in vivo, data from cynomolgus monkey toxicology studies with 2 bispecific T-cell engager molecules targeting FLT3 were leveraged; findings were consistent with the targeted killing of bone marrow cells expressing cell surface FLT3. Potential AMG 553-induced cytotoxicity was assessed against a wide range of normal human primary cells and cell lines; cytotoxicity was observed against FLT3-positive AML cell lines and a percentage of primary bone marrow CD34+ cells. In conclusion, the nonclinical safety data suggest that AMG 553 can target FLT3 protein on AML cells, whereas only affecting a percentage of normal hematopoietic stem and progenitor cells, supporting clinical development.

Characterization of a Novel FLT3 BiTE Molecule for the Treatment of Acute Myeloid Leukemia.

Despite advances in the treatment of acute myeloid leukemia (AML), novel therapies are needed to induce deeper and more durable clinical response. Bispecific T-cell Engager (BiTE) molecules, which redirect patient T cells to lyse tumor cells, are a clinically validated modality for hematologic malignancies. Due to broad AML expression and limited normal tissue expression, fms-related tyrosine kinase 3 (FLT3) is proposed to be an optimal BiTE molecule target. Expression profiling of FLT3 was performed in primary AML patient samples and normal hematopoietic cells and nonhematopoietic tissues. Two novel FLT3 BiTE molecules, one with a half-life extending (HLE) Fc moiety and one without, were assessed for T-cell-dependent cellular cytotoxicity (TDCC) of FLT3-positive cell lines in vitro, in vivo, and ex vivo FLT3 protein was detected on the surface of most primary AML bulk and leukemic stem cells but only a fraction of normal hematopoietic stem and progenitor cells. FLT3 protein detected in nonhematopoietic cells was cytoplasmic. FLT3 BiTE molecules induced TDCC of FLT3-positive cells in vitro, reduced tumor growth and increased survival in AML mouse models in vivo Both molecules exhibited reproducible pharmacokinetic and pharmacodynamic profiles in cynomolgus monkeys in vivo, including elimination of FLT3-positive cells in blood and bone marrow. In ex vivo cultures of primary AML samples, patient T cells induced TDCC of FLT3-positive target cells. Combination with PD-1 blockade increased BiTE activity. These data support the clinical development of an FLT3 targeting BiTE molecule for the treatment of AML.

Dendritic cell-derived hepcidin sequesters iron from the microbiota to promote mucosal healing.

Bleeding and altered iron distribution occur in multiple gastrointestinal diseases, but the importance and regulation of these changes remain unclear. We found that hepcidin, the master regulator of systemic iron homeostasis, is required for tissue repair in the mouse intestine after experimental damage. This effect was independent of hepatocyte-derived hepcidin or systemic iron levels. Rather, we identified conventional dendritic cells (cDCs) as a source of hepcidin that is induced by microbial stimulation in mice, prominent in the inflamed intestine of humans, and essential for tissue repair. cDC-derived hepcidin acted on ferroportin-expressing phagocytes to promote local iron sequestration, which regulated the microbiota and consequently facilitated intestinal repair. Collectively, these results identify a pathway whereby cDC-derived hepcidin promotes mucosal healing in the intestine through means of nutritional immunity.

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Discovery of N-(1-Acryloylazetidin-3-yl)-2-(1H-indol-1-yl)acetamides as Covalent Inhibitors of KRASG12C.

KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C, which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRASG12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRASG12C with submicromolar inhibition of downstream signaling in a KRASG12C-specific manner.

Classical and intermediate monocytes scavenge non-transferrin-bound iron and damaged erythrocytes.

Myelomonocytic cells are critically involved in iron turnover as aged RBC recyclers. Human monocytes are divided in 3 subpopulations of classical, intermediate, and nonclassical cells, differing in inflammatory and migratory phenotype. Their functions in iron homeostasis are, however, unclear. Here, we asked whether the functional diversity of monocyte subsets translates into differences in handling physiological and pathological iron species. By microarray data analysis and flow cytometry we identified a set of iron-related genes and proteins upregulated in classical and, in part, intermediate monocytes. These included the iron exporter ferroportin (FPN1), ferritin, transferrin receptor, putative transporters of non-transferrin-bound iron (NTBI), and receptors for damaged erythrocytes. Consequently, classical monocytes displayed superior scavenging capabilities of potentially toxic NTBI, which were augmented by blocking iron export via hepcidin. The same subset and, to a lesser extent, the intermediate population, efficiently cleared damaged erythrocytes in vitro and mediated erythrophagocytosis in vivo in healthy volunteers and patients having received blood transfusions. To summarize, our data underline the physiologically important function of the classical and intermediate subset in clearing NTBI and damaged RBCs. As such, these cells may play a nonnegligible role in iron homeostasis and limit iron toxicity in iron overload conditions.

Identification of Antibody and Small Molecule Antagonists of Ferroportin-Hepcidin Interaction.

The iron exporter ferroportin and its ligand, the hormone hepcidin, control fluxes of stored and recycled iron for use in a variety of essential biochemical processes. Inflammatory disorders and malignancies are often associated with high hepcidin levels, leading to ferroportin down-regulation, iron sequestration in tissue macrophages and subsequent anemia. The objective of this research was to develop reagents to characterize the expression of ferroportin, the interaction between ferroportin and hepcidin, as well as to identify novel ferroportin antagonists capable of maintaining iron export in the presence of hepcidin. Development of investigative tools that enabled cell-based screening assays is described in detail, including specific and sensitive monoclonal antibodies that detect endogenously-expressed human and mouse ferroportin and fluorescently-labeled chemically-synthesized human hepcidin. Large and small molecule antagonists inhibiting hepcidin-mediated ferroportin internalization were identified, and unique insights into the requirements for interaction between these two key iron homeostasis molecules are provided.

Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing.

For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis.

On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver.

Iron is an essential component of the erythrocyte protein hemoglobin and is crucial to oxygen transport in vertebrates. In the steady state, erythrocyte production is in equilibrium with erythrocyte removal. In various pathophysiological conditions, however, erythrocyte life span is compromised severely, which threatens the organism with anemia and iron toxicity. Here we identify an on-demand mechanism that clears erythrocytes and recycles iron. We show that monocytes that express high levels of lymphocyte antigen 6 complex, locus C1 (LY6C1, also known as Ly-6C) ingest stressed and senescent erythrocytes, accumulate in the liver via coordinated chemotactic cues, and differentiate into ferroportin 1 (FPN1, encoded by SLC40A1)-expressing macrophages that can deliver iron to hepatocytes. Monocyte-derived FPN1(+)Tim-4(neg) macrophages are transient, reside alongside embryonically derived T cell immunoglobulin and mucin domain containing 4 (Timd4, also known as Tim-4)(high) Kupffer cells (KCs), and depend on the growth factor Csf1 and the transcription factor Nrf2 (encoded by Nfe2l2). The spleen, likewise, recruits iron-loaded Ly-6C(high) monocytes, but these do not differentiate into iron-recycling macrophages, owing to the suppressive action of Csf2. The accumulation of a transient macrophage population in the liver also occurs in mouse models of hemolytic anemia, anemia of inflammation, and sickle cell disease. Inhibition of monocyte recruitment to the liver during stressed erythrocyte delivery leads to kidney and liver damage. These observations identify the liver as the primary organ that supports rapid erythrocyte removal and iron recycling, and uncover a mechanism by which the body adapts to fluctuations in erythrocyte integrity.

High-Throughput Mass Spectrometric Analysis of Covalent Protein-Inhibitor Adducts for the Discovery of Irreversible Inhibitors: A Complete Workflow.

We have implemented a solid-phase extraction based time-of-flight mass spectrometer system in combination with novel informatics to rapidly screen and characterize the covalent binding of different irreversible inhibitors to intact proteins. This high-throughput screening platform can be used to accurately detect and quantitate the extent of formation of different covalent protein-inhibitor adducts between electrophilic inhibitors and nucleophilic residues such as cysteine or lysine. For a representative 19.5 kDa protein, the analysis time is approximately 20 s per sample, including an efficient sample loading and desalting step. Accurate protein masses are measured (±0.5 amu of the theoretical molecular weight; measured precision of ±0.02 amu). The fraction of protein reacted with an electrophilic compound is determined relative to an unmodified protein control. A key element of the workflow is the automated identification and quantitation of the expected masses of covalent protein-inhibitor adducts using a custom routine that obviates the need to manually inspect each individual spectrum. Parallel screens were performed on a library of approximately 1000 acrylamide containing compounds (different structures and reactivities) using the solid-phase extraction mass spectrometry based assay and a fluorescence based thiol-reactive probe assay enabling comparison of false positives and false negatives between these orthogonal screening approaches.

Systematic Study of the Glutathione (GSH) Reactivity of N-Arylacrylamides: 1. Effects of Aryl Substitution.

Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relationships among N-arylacrylamides, we determined glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. We find that substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. In addition, we note a correlation between (1)H and (13)C NMR chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic candidates in a prospective fashion.

Design Rationale and Development Approach for Pegfilgrastim as a Long-Acting Granulocyte Colony-Stimulating Factor.

Filgrastim, a recombinant methionyl human granulocyte colony-stimulating factor (G-CSF) (r-metHuG-CSF), is efficacious in stimulating neutrophil production and maturation to prevent febrile neutropenia (FN) in response to chemotherapy. Because of its relatively short circulating half-life, daily filgrastim injections are required to stimulate neutrophil recovery. In an effort to develop a long-acting form of filgrastim that was as safe and efficacious as filgrastim but had a longer in vivo residence time, a number of strategies were considered. Ultimately, fusion of filgrastim to polyethylene glycol (PEG) was selected. Following extensive analysis of conjugation chemistries as well as in vitro and in vivo characterization of a panel of PEGylated proteins, a construct containing a 20 kDa PEG moiety covalently conjugated to the N-terminus of filgrastim was chosen for advancement as pegfilgrastim. Pegfilgrastim is primarily cleared by neutrophils and neutrophil precursors (rather than the kidneys), meaning that clearance from the circulation is self-regulating and pegfilgrastim is eliminated only after neutrophils start to recover. Importantly, addition of PEG did not alter the mechanism of action and safety profile compared to filgrastim. Clinical evaluation revealed that a single 6 mg dose effectively reduces the duration of neutropenia and risk of FN in patients receiving chemotherapy. This work demonstrates the benefit of using PEGylation to generate pegfilgrastim, which allows for once-per-chemotherapy cycle administration while maintaining similar safety and efficacy profiles as those for multiple daily administration of filgrastim. Approaches that may provide advances for therapeutic agonists of G-CSF receptor are also discussed.

Hepcidin regulation in prostate and its disruption in prostate cancer.

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression.

IL-22 regulates iron availability in vivo through the induction of hepcidin.

Iron is a trace element important for the proper folding and function of various proteins. Physiological regulation of iron stores is of critical importance for RBC production and antimicrobial defense. Hepcidin is a key regulator of iron levels within the body. Under conditions of iron deficiency, hepcidin expression is reduced to promote increased iron uptake from the diet and release from cells, whereas during conditions of iron excess, induction of hepcidin restricts iron uptake and movement within the body. The cytokine IL-6 is well established as an important inducer of hepcidin. The presence of this cytokine during inflammatory states can induce hepcidin production, iron deficiency, and anemia. In this study, we show that IL-22 also influences hepcidin production in vivo. Injection of mice with exogenous mouse IgG1 Fc fused to the N terminus of mouse IL-22 (Fc-IL-22), an IL-22R agonist with prolonged and enhanced functional potency, induced hepcidin production, with a subsequent decrease in circulating serum iron and hemoglobin levels and a concomitant increase in iron accumulation within the spleen. This response was independent of IL-6 and was attenuated in the absence of the IL-22R-associated signaling kinase, Tyk2. Ab-mediated blockade of hepcidin partially reversed the effects on iron biology caused by IL-22R stimulation. Taken together, these data suggest that exogenous IL-22 regulates hepcidin production to physiologically influence iron usage.

Molecular mechanism of hepcidin-mediated ferroportin internalization requires ferroportin lysines, not tyrosines or JAK-STAT.

Ferroportin is the primary means of cellular iron efflux and a key component of iron metabolism. Hepcidin regulates Fpn activity by inducing its internalization and degradation. The mechanism of internalization is reported to require JAK2 activation, phosphorylation of Fpn tyrosine residues 302 and 303, and initiation of transcription through STAT3 phosphorylation. These findings suggest Fpn may be a target for therapeutic intervention through JAK2 modulation. To evaluate the proposed mechanism, Fpn internalization was assessed using several techniques combined with reagents that specifically recognized cell-surface Fpn. In vitro results demonstrated that Hepc-induced Fpn internalization did not require JAK2 or phosphorylation of Fpn residues 302 and 303, nor did it induce JAK-STAT signaling. In vivo, inhibition of JAK2 had no effect on Hepc-induced hypoferremia. However, internalization was delayed by mutation of two Fpn lysine residues that may be targets of ubiquitination.