Inhibitors targeting protein-protein interactions

Adrian Whitty, Karen Allen, John Porco, and Sandor Vajda.

Supported by:
NIH R01 GM117350 “Molecular Mechanism Of The Nfkappab Essential Modulator (Nemo) Scaffold Protein Mutated In Human Immunodeficiencies” (PI: Adrian Whitty)
NIH R35 GM118078 “Analysis and Prediction of Molecular Interactions” (PI: Sandor Vajda)

NF-κB essential modulator (NEMO) is a component of the Inhibitor of κB (I-κB) kinase (IKK) complex, which additionally contains IKKα and IKKβ, is an essential component of the canonical NF-κB signaling pathway. NF-κB signaling is widely studied due to its involvement in critical cellular processes, including cell survival, proliferation and differentiation, and dysregulation of the pathway has been linked to a variety of inflammatory diseases and some cancers. NF-κB signaling is activated by a wide range of stimuli, including pro-inflammatory cytokines and other receptor mediated signals. These signals lead to formation and activation of the IKK complex, which phosphorylates I-κB causing it to dissociate from its latent complex with NF-κB, thereby releasing NF-κB to translocate to the nucleus where it mediates target gene transcription. IKK is therefore crucial to activation of NF-κB signaling, and inhibition of IKK or disruption of the NEMO-IKKα/β interaction that holds the complex together has emerged as a promising therapeutic target.

Our collaborative research focuses on the interactions between NEMO and IKK with the goal of identifying small molecule antagonists that can prevent the formation of their complex. The development of “drug-like” small molecule inhibitors against protein-protein interaction (PPI) interfaces presents a particular challenge for contemporary drug discovery. Addressing this multi-faceted problem consequently requires a multidisciplinary approach that brings to bear sound structural and biochemical methods together with innovative chemistry that right from the start is guided jointly by considerations of activity against the target and also of optimizing ADME properties. The multi-disciplinary team we have assembled possesses strong expertise and highly relevant experience across all of these areas.

Based on theoretical and practical experience with PPI targets we pursue two complementary approaches as the best for generating novel chemical structures as inhibitors of NEMO/IKKβ binding:

  1. A fragment-based approach, enhanced by guidance from analysis of the physicochemical composition of PPI interfaces and by the FTMap computational solvent mapping algorithm, developed in the Vajda lab, to identify specific chemical functionalities that interact favorably with the binding hotspot in the NEMO N-terminal domain.
  2. A systematic exploration of natural product-inspired synthetic macrocycles, synthesized in the Porco group,  with functionality biased towards likely PPI binders (from FTMap results and structural analysis of PPI interfaces), with parallel screening for the key predictive in vitro ADME properties of solubility and cell permeability.
Fig. 1 Workflow
Fig. 1 Workflow

We have recently combined computational and experimental methods to determine the contributions of individual amino acid residues of IKKβ to the free energy of binding between  NEMO and IKKβ (Golden et al., J Am Chem Soc. 135(16):6242-56). The collaboration also includes the analysis of NEMO using Small Angle X-ray Scattering (SACS) and determining the X-ray structure of NEMO. Some of the steps of this work are shown in Fig. 1. Initial screening and hit-to-lead chemistry, supported by extensive structural and biochemical validation, will lead to a decision as to which compound series have the best prospects for success based on a comprehensive evaluation of their potency, selectivity and in vitro ADME properties. Subsequent efforts will focus on optimizing the selected lead series, and on characterizing its interactions with NEMO using a variety of structural, biochemical and biophysical methods to understand the structural and physico-chemical basis for its activity.

Trilles R, Beglov D, Chen Q, He H, Wireman R, Reed A, Chennamadhavuni S, Panek JS, Brown LE, Vajda S, Porco JA Jr, Kelley MR, Georgiadis MM. Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1. Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1. J Med Chem. 2019 Feb 28;62(4):1971-1988. doi: 10.1021/acs.jmedchem.8b01529.

Lukose V, Luo L, Kozakov D, Vajda S, Allen KN, Imperiali B. Conservation and Covariance in Small Bacterial Phosphoglycosyltransferases Identify the Functional Catalytic Core. Biochemistry. 2015 Dec 22;54(50):7326-34. doi: 10.1021/acs.biochem.5b01086.

Villar EA, Beglov D, Chennamadhavuni S, Porco JA Jr, Kozakov D, Vajda S, Whitty A. How proteins bind macrocycles. Nat Chem Biol. 2014 Sep;10(9):723-31. doi: 10.1038/nchembio.1584.

Golden MS, Cote SM, Sayeg M, Zerbe BS, Villar EA, Beglov D, Sazinsky SL, Georgiadis RM, Vajda S, Kozakov D, Whitty A. Comprehensive experimental and computational analysis of binding energy hot spots at the NF-κB essential modulator/IKKβ protein-protein interface. J Am Chem Soc. 2013 Apr 24;135(16):6242-56. doi: 10.1021/ja400914z.