NEMO (NF-κB essential modulator) associates with the catalytic subunits IKKα and IKKβ to form the IκB kinase (IKK) complex, and is a key regulator of NF-κB pathway signaling. Biochemical and structural characterization of NEMO has been challenging, however, leading to conflicting data on basic biochemical properties such as the oligomeric state of active NEMO and its binding affinity for IKKβ. We show that up to seven of NEMO’s 11 cysteine residues can be mutated to generate recombinant full-length NEMO that is highly soluble and active. Using a fluorescence anisotropy binding assay we show that full-length NEMO binds a 44-mer peptide encompassing residues 701-745 of IKKβ with KD = 2.2 ± 0.8 nM. The IKKβ binding affinities of mutants with five and seven Cys-to-Ala substitutions are indistinguishable from that of wild-type NEMO. Moreover, when expressed in NEMO −/− fibroblasts, the 5xAla and 7xAla NEMO mutants can interact with cellular IKKβ and restore NF-κB signaling to provide protection against TNFα-induced cell death. Treatment of the NEMO-reconstituted cells with H2O2 led to formation of covalent dimers for wild-type NEMO and the 5xAla mutant, but not for the 7xAla mutant, confirming that Cys54 and/or Cys347 can mediate inter-chain disulfide bonding. However, the IKKβ binding affinity of NEMO is unaffected by the presence or absence of inter-chain disulfide bonding at Cys54 – which lies within the IKKβ binding domain of NEMO – or at Cys347, indicating that NEMO exists as a noncovalent dimer independent of the redox state of its cysteines. This conclusion was corroborated by the observation that the secondary structure content of NEMO and its thermal stability were independent of the presence or absence of inter-chain disulfide bonds.

We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between NF-κB Essential Modulator (NEMO) and IκB kinase subunit β (IKKβ), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NBD region of IKKβ contains the highest concentration of hot spot residues, the strongest of which are W739, W741 and L742 (ΔΔG = 4.3, 3.5 and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKβ L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot spot regions centered on IKKβ residues L708/V709 and L719/I723. The computational approach successfully identified all three hot spot regions on IKKβ. Moreover, the method was able to accurately quantify the energetic importance of all hot spots residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small molecule inhibitors that target the NEMO/IKKβ interaction. They additionally clarify the structural and energetic complementarity between “pocket-forming” and “pocket occupying” hot spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of α-motor neurons, leading to profound skeletal muscle atrophy. Patients also suffer from decreased bone mineral density and increased fracture risk. The majority of treatments for SMA, approved or in clinic trials, focus on addressing the underlying cause of disease, insufficient production of full-length SMN protein. While restoration of SMN has resulted in improvements in functional measures, significant deficits remain in both mice and SMA patients following treatment. Motor function in SMA patients may be additionally improved by targeting skeletal muscle to reduce atrophy and improve muscle strength. Inhibition of myostatin, a negative regulator of muscle mass, offers a promising approach to increase muscle function in SMA patients. Here we demonstrate that muSRK-015P, a monoclonal antibody which specifically inhibits myostatin activation, effectively increases muscle mass and function in two variants of the pharmacological mouse model of SMA in which pharmacologic restoration of SMN has taken place either 1 or 24 days after birth to reflect early or later therapeutic intervention. Additionally, muSRK-015P treatment improves the cortical and trabecular bone phenotypes in these mice. These data indicate that preventing myostatin activation has therapeutic potential in addressing muscle and bone deficiencies in SMA patients. An optimized variant of SRK-015P, SRK-015, is currently in clinical development for treatment of SMA.

We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity.

Introduction: Apitegromab (SRK-015) is an anti-promyostatin monoclonal antibody under development to improve motor function in patients with spinal muscular atrophy, a rare neuromuscular disease. This phase 1 doubleblind, placebo-controlled study assessed safety, pharmacokinetic parameters, pharmacodynamics (serum latent myostatin), and immunogenicity of single and multiple ascending doses of apitegromab in healthy adult subjects. Methods: Subjects were administered single intravenous ascending doses of apitegromab of 1, 3, 10, 20, 30 mg/kg or placebo, and multiple intravenous ascending doses of apitegromab of 10, 20, 30 mg/kg or placebo. Results: Following single ascending doses, the pharmacokinetic parameters of apitegromab appeared to be similar across all dose groups, following a biphasic pattern of decline in the concentration-time curve. The mean apparent terminal t 1/2 after single intravenous doses of apitegromab ranged from 24 to 31 days across dose groups. Dose-related increases were observed in C max following multiple ascending doses. Single and multiple apitegromab doses resulted in dose-dependent and sustained increases in serum latent myostatin, indicating robust target engagement. Apitegromab was safe and well tolerated, on the basis of the adverse event (AE) profile with no clinically meaningful changes in baseline vital signs, electrocardiograms, or clinical laboratory parameters and no anti-drug antibody formation. Conclusion: These results support continued investigation of apitegromab for the treatment of patients with milder forms (type 2 and 3) of spinal muscular atrophy.