PPI Targeted Libraries by Receptor-based Approach

The importance of the complex network of direct interactions between proteins — known as the interactome - to both biological systems and the development of diseases is widely recognized. Despite this, small-molecule drugs that act by directly disrupting the interaction between two proteins are relatively rare in comparison to other drug classes, and protein-protein interactions (PPIs) are viewed as challenging, in some cases essentially “undruggable” targets. However, recent researchers’ reports have shown that certain classes of PPI are amenable to small-molecule inhibition. Typically, these PPI inhibitors disrupt the interaction between a globular protein and a single peptide chain on the partner protein by binding into pockets on the surface of the globular protein. PPIs can be classified into groups based on common structural elements in both the globular protein and the peptide chain. Most notably, the presence of secondary structural features within the peptide chain, such as α-helices and β-strands, has significant ramifications for the design of inhibitors that function by mimicking and displacing these peptides. [1]

Life Chemicals has designed PPI Screening Libraries by a receptor-based approach that gathered a number of the most attractive PPI targets for therapeutic intervention via disrupting specific functional interactions between proteins. These include bromodomains, integrins, apoptosis regulators, and oncoproteins that belong to different PPI types and have been proven to be druggable by small-molecule compounds.

Presented here is a compound collection of potential PPI modulators that contains over 8,700 drug-like screening compounds specifically targeting PPIs. The approach used to design the Libraries is based on flexible molecular docking of the Life Chemicals HTS Compound Collection into a PPI interface of each drug target in order to predict molecules with the highest affinity, and, hence, the capability to prevent interaction with the binding partner. Several docking constraints (positional, H-bond, metal chelation) and amino acid rotatable groups involved in protein-ligand interaction are allowed where reasonable. 

To cover relevant chemical space that includes peptidomimetic molecules, the Library was not made compliant with Lipinski's Rule of Five. Each Library contains compounds free of PAINS, toxic, and reactive groups.

BCL‑2

The BCL-2 family of proteins is pivotal in regulating cell death through the control of the integrity of the outer mitochondrial membrane. Pro-apoptotic BCL-2 family of proteins, such as BAK and BAX, have an essential role in apoptosis. The effect of these proteins is blocked when they are sequestered by anti-apoptotic binding partners such as BCL-2 and BCL-XL. Small molecules that disrupt this interaction by binding to the anti-apoptotic BCL-2 family proteins have been designed to induce apoptosis of cancer cells [2].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 6GL8
  • Positional constraints: yes
  • H-bond constraints: ALA149, GLU136, GLN118, ASP111
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 372

 Examples of virtual hit compounds (binding site: 6GL8) from the BCL-2 compound set.

Fig. 1. Examples of virtual hit compounds (binding site: 6GL8) from the BCL-2 compound set.

MDM2–p53

p53 is a potent tumor suppressor and is an attractive cancer therapeutic target because it can be functionally activated to eradicate tumors. The gene encoding p53 protein is mutated or deleted in half of the human cancers, which inactivates tumor suppressor activity. In the remaining cancers with wild-type p53 status, its function is effectively inhibited through direct interaction with the human murine double minute 2 (MDM2) oncoprotein. Blocking the MDM2-p53 interaction to reactivate the p53 function is a promising cancer therapeutic strategy [3].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 4ZYF
  • Positional constraints: yes
  • H-bond constraints: HOH323, HOH302, VAL93, GLN72
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,680

Examples of virtual hit compounds (binding site: 4ZYF) from the MDM2–p53 library.

Fig. 2. Examples of virtual hit compounds (binding site: 4ZYF) from the MDM2–p53 library.

LFA1–ICAM1

The lymphocyte function-associated antigen-1 (LFA-1) (also known as CD11a/CD18 and αLβ2) is just one of many integrins in the human body, but its significance is derived from its exclusive presence in leukocytes. Many studies have shown the relation of LFA-1 and its primary ligand ICAM-1 (or CD54) with cancer through the function of lymphocytes and myeloid cells on tumor cells. LFA-1 mediates the interaction of leukocytes with tumors and ICAM-1 has a vital role in tumor dynamics, which can be independent of its interaction with LFA-1 [4].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 3M6F, 2ICA
  • Positional constraints: no
  • H-bond constraints: HOH408, HOH324
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,779

Examples of virtual hit compounds (binding site: 3M6F) from the LFA1–ICAM1 library.

Fig. 3. Examples of virtual hit compounds (binding site: 3M6F) from the LFA1–ICAM1 library.

αIIbβ3

The integrin αIIbβ3 on platelets is activated and binds to its ligand fibrinogen at the early stages of hemostasis and thrombosis. The integrin αIIbβ3 binds specifically to the distal ends of the dimeric fibrinogen molecule in a natively unstructured region at the C terminus of the γ subunit (γC peptide). The large separation of ∼440 Å between the two αIIbβ3-binding sites on fibrinogen is well suited for cross-linking of platelets, which results in platelet aggregation and formation of platelet plugs in hemostasis and thrombosis. Further on during hemostasis, cleavage of peptides from the N termini of the fibrinogen α and β subunits stimulates an assembly of fibrinogen into fibrin. Yet later, adjacent fibrinogen molecules within fibrin are cross-linked through their γC peptides by the factor XIIIa transglutaminase. Due to biological and clinical importance of the γC peptide in hemostasis and thrombosis, there has been a significant interest in determining its biologically relevant integrin-bound conformation. Two drugs, a small molecule (tirofiban) and a cyclic peptide (eptifibatide), are currently used clinically to prevent thrombosis. Their complex structures with αIIbβ3 have been determined [5].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 2VDM
  • Positional constraints: no
  • H-bond constraints: TYR122, SER123, ASN215, ASN215, HOH4069, SER225, ASP224
  • Metal coordination: yes 
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,445

Examples of virtual hit compounds (binding site: 2VDM) from the αIIbβ3 library.

Fig. 4. Examples of virtual hit compounds (binding site: 2VDM) from the αIIbβ3 library. 

IAP

Members of the mammalian inhibitor of apoptosis (IAP) family of proteins, including X chromosome-linked IAP (XIAP), cellular IAP 1 (cIAP1), cellular IAP 2 (cIAP2), and melanoma IAP (ML-IAP), are frequently overexpressed in cancer cells, where they confer protection against a variety of pro-apoptotic stimuli. The IAP proteins have also been demonstrated to function in the regulation of signal transduction pathways associated with malignancy. Efforts to target the IAP proteins have focused on the design of small molecules that mimic the binding of the endogenous IAP antagonist second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI (Smac/DIABLO) to a shallow groove on the surface of select IAP baculoviral IAP repeat (BIR) domains [6]. 

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 3UW5
  • Positional constraints: no
  • H-bond constraints: ASP138, GLN132, GLN132, TRP147
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 661

Examples of virtual hit compounds (binding site: 3UW5) from the IAP library.

 

Fig. 5. Examples of virtual hit compounds (binding site: 3UW5) from the IAP library.

BET Bromodomain

Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that comprise the ubiquitously expressed BRD2, BRD3, BRD4, as well as BRDT. BET proteins also recognize acetylated non-histone proteins, including different transcription factors. In addition, BET can also have kinase activity, a function not yet fully understood. Several lines of evidence coming from preclinical studies indicate a role of BET proteins in human cancer and have provided the rationale for targeting BET proteins as a strategy for the development of new anticancer drugs. Genetic screening programs performed for different tumor types have recurrently identified the genes encoding BET proteins as those on which neoplastic cells depend for their survival [7].

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 4J1P
  • Positional constraints: no
  • H-bond constraints: HOH604, ASN429
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,573

 

 Examples of virtual hit compounds (binding site: 4J1P) from the BET Bromodomain library.

Fig. 6. Examples of virtual hit compounds (binding site: 4J1P) from the BET Bromodomain library.

BRD9

Bromodomains (BRDs) are protein interaction modules that selectively recognize ε-N-lysine acetylation motifs, a key event in reading the posttranslational modifications that constitute the epigenetic code. The BRD-containing protein 7 (BRD7), which is frequently down-regulated in cancer, has a proposed tumor suppression function through the regulation of p53 and PI3K. Furthermore, BRD7 has been shown to be required for BRCA1-dependent transcription, and BRD7 polymorphism has been linked to an increased risk of pancreatic cancer. In contrast, BRD9 is often over-expressed in cancer owing to a gain of the short arm of chromosome 5 (5p), the most frequent karyotypic change in cervical cancer. The closely related BRDs BRD7 and BRD9 are a part of the SWI/SNF nucleosome remodeling complex, which plays a crucial role in regulating gene expression programs, including the expression of inflammatory genes. Owing to the complexity of BRD7/9-mediated interactions in chromatin, selective potent inhibitors of these bromodomains would constitute valuable biological tools, enabling functional studies on these essential chromatin interaction domains and potentially allowing for exploitation in small-molecule therapies for various diseases [8]. 

Key features

  • Method: high-throughput virtual screening (docking)
  • X-Ray data used: 5EU1
  • Positional constraints: no
  • H-bond constraints: HOH301, ASN100
  • Protein rotatable groups allowed: yes
  • Filters used: PAINS, toxic, reactive
  • Number of compounds selected: 1,563

 Examples of virtual hit compounds (binding site: 5EU1) from the BRD9 library.

Fig. 7. Examples of virtual hit compounds (binding site: 5EU1) from the BRD9 library.

References:

  1. Scott DE, Bayly AR, Abell C, Skidmore J. Small molecules, big targets: drug discovery faces the protein-protein interaction challenge. Nat Rev Drug Discov. 2016;15(8):533-550. doi:10.1038/nrd.2016.29
  2. Vogler, M., Dinsdale, D., Dyer, M. J. & Cohen, G. M. Bcl‑2 inhibitors: small molecules with a big impact on cancer therapy. Cell Death Differ. 16, 360–367 (2009).
  3. Shangary S, Wang S. Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res. 2008;14(17):5318-5324. doi:10.1158/1078-0432.CCR-07-5136
  4. Reina M, Espel E. Role of LFA-1 and ICAM-1 in Cancer. Cancers (Basel).2017;9(11):153. doi:10.3390/cancers9110153
  5. Springer TA, Zhu J, Xiao T. Structural basis for distinctive recognition of fibrinogen gammaC peptide by the platelet integrin alphaIIbbeta3. J Cell Biol.2008;182(4):791-800. doi:10.1083/jcb.200801146
  6. Flygare JA, Beresini M, Budha N, et al. Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152). J Med Chem.2012;55(9):4101-4113. doi:10.1021/jm300060k
  7. Stathis A, Bertoni F. BET Proteins as Targets for Anticancer Treatment. Cancer Discov.2018;8(1):24-36. doi:10.1158/2159-8290.CD-17-0605
  8. Clark PG, Vieira LC, Tallant C, et al. LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor. Angew Chem Int Ed Engl. 2015;54(21):6217-6221. doi:10.1002/anie.201501394
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