Targeting the γ-Aminobutyric Acid Type B (GABA_B) Receptor Complex: Development of Inhibitors Targeting the K⁺ Channel Tetramerization Domain (KCTD) Containing Proteins/GABA_B Receptor Protein–Protein Interaction

Abstract

Targeting multiprotein receptor complexes, rather than receptors directly, is a promising concept in drug discovery. This is particularly relevant to the GABAB receptor complex, which plays a prominent role in many brain functions and diseases. Here, we provide the first studies targeting a key protein–protein interaction of the GABAB receptor complexthe interaction with KCTD proteins. By employing the μSPOT technology, we first defined the GABAB receptor-binding epitope mediating the KCTD interaction. Subsequently, we developed a highly potent peptide-based inhibitor that interferes with the KCTD/GABAB receptor complex and efficiently isolates endogenous KCTD proteins from mouse brain lysates. X-ray crystallography and SEC-MALS revealed inhibitor induced oligomerization of KCTD16 into a distinct hexameric structure. Thus, we provide a template for modulating the GABAB receptor complex, revealing a fundamentally novel approach for targeting GABAB receptor-associated neuropsychiatric disorders.

Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases

“…Unfortunately, the crude peptides could not be triturated before chromatography due to the minute amounts produced and the resulting HPLC traces therefore included protecting group byproducts. Nevertheless, an average purity of 41%, corresponding to average coupling efficiency of at least 95%, was observed, which is in line with standard SPOT [42][43] and µSPOT 44 peptide synthesis. The detection of a dilution series of His-tagged HDAC2 spotted on a µSPOT slide indicated a linear correlation between signal and spotted amount (Supplementary Fig.…”

AlphaFold-Predicted Structures of KCTD Proteins Unravel Previously Undetected Relationships among the Members of the Family

“…The structure of the BTB domain, as an isolated entity, has been reported for KCTD1 (PDB entries 5BXD and 5BXB), KCTD5 (PDB entry 3DRZ), KCTD10 (5FTA), KCTD13 (4UIJ), KCTD16 (5A15, 6OCR, 6I0Q, and 6OCT), KCTD17 (5A6R), and SHKBP1 (4CRH). These structures have highlighted the versatility of this domain [ 15 ], which was also corroborated by molecular dynamics simulations [ 18 ], to oligomerize in different oligomeric states that include monomeric, tetrameric, open/close pentameric, and hexameric assemblies [ 15 , 17 , 34 ]. This domain has also been characterized in complex with a peptide of the GABAB2 receptor (6OCP and 6M8R) [ 22 , 35 ].…”

Low-cost synthesis of peptide libraries and their use for binding studies via temperature-related intensity change

“…To assess the peptide purity after synthesis, which may vary depending on peptide length and sequence, we recommend conducting liquid chromatography coupled to mass spectrometry (LC-MS) measurements of control peptides ( Schulte et al., 2020 , Moreno-Yruela et al., 2020 , Sereikaite et al., 2019 ) (see 18). Alternatively, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry can be performed to determine peptide purity.…”

“…Affinity data obtained with peptides of largely differing length can be expected to be biased by variations in amounts and purities. In this line, we recommend an upper limit of 30 aas for affinity determination, since a commonly observed coupling efficiency of 93%–98% corresponds to a peptide purity of 11%–55% ( Schulte et al., 2020 , Moreno-Yruela et al., 2020 , Sereikaite et al., 2019 ) . Generally, representative quality controls should always be included in any peptide library to assess the peptide purity (see Quality Control by LC-MS).…”