Fragment-based drug discovery and protein–protein interactions

Figures & data

Figure 1 Orthosteric inhibition of a protein–protein interaction versus allosteric inhibition.

Notes: An orthosteric inhibitor (represented as a purple rectangle) interferes directly with the protein–protein interface, disrupting binding, whilst an allosteric inhibitor (represented as a green triangle) induces a conformational change to the binding interface region of the protein that indirectly disables binding.

Figure 2 Protein–protein interaction hot spots and allosteric sites.

Notes: (A) Protein–protein interface of the third baculoviral IAP repeat (BIR3) of XIAP and caspase-9 (PDB code = 1NW9), with hot spots computed by the HotRegion webserver (http://prism.ccbb.ku.edu.tr/hotregion/) colored red against the otherwise grey surface. The cartoon representations of BIR3-XIAP and caspase-9 are colored gray and purple, respectively. (B) The allosteric site of HCV NS3 (PDB code = 1CU1) is shown as a molecular surface and colored orange. The protease domain, the NS4a cofactor, and the helicase domain are highlighted in green, magenta, and cyan, respectively. The allosteric site is located at the interface between the protease and helicase domains. Figure prepared using PyMOL Molecular Graphics System, Version 1.2r2; Schrödinger, LLC, Camberley, UK.
Abbreviations: HCV, hepatitis C virus; IAP, inhibitors of apoptosis; PDB, Protein Data Bank.

Figure 3 Summary of selected properties and characteristics of fragment hits against PPI targets compared with fragment hits against typical enzyme targets (Std).

Notes: All descriptors were computed using Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group Inc., Montreal, Canada. (A) Acid, base, and hydrophobicity statistics for the two sets of fragment hits. Hydrophobic proportion of molecules was calculated as (hydrophobic atom count)/(heavy atom count). (B) Mean statistics over both datasets of fragments including MWT (Da), logP, molecular globularity, normalized principal moments of inertia ratios (NPR1 and NPR2), TPSA (Ų), number of rotatable bonds, LE, and LLE. (C) Histogram plot (y-axis is percentage of dataset) showing MWT (Da) distribution across both sets of fragments. The blue line represents the PPI fragment hits, and the red line shows the non-PPI, standard fragment hits. (D) Histogram plot (y-axis is percentage of dataset) showing logP distribution across both sets of fragments. The blue line represents the PPI fragment hits, and the red line shows the non-PPI, standard fragment hits. (E) Histogram plot (y-axis is percentage of dataset) showing molecular globularity distribution across both sets of fragments. The blue line represents the PPI fragment hits, and the red line shows the non-PPI, standard fragment hits. (F) Normalized principal moments of inertia ratios plot (NPR1 versus NPR2) showing a measure of the three-dimensional character of both datasets. Blue dots represent the PPI fragment hits, and red dots represent the non-PPI fragment hits. Disk-like structures should cluster towards the bottom apex of the triangular plot, rod-like structures towards the top left and spherical structures to the top right.
Abbreviations: LE, ligand efficiency; LLE, lipophilic ligand efficiency; MWT, molecular weight; PPI, protein–protein interaction; Std, standard; TPSA, topological polar surface area.

Table 1 Major screening and validation techniques used to detect PPI fragment hits

Technique	Application	Type of information	Case studies
NMR
Protein observed     2D-HSQC     2D-HMQC	Primary screening Competitive PPI assay Hit validation	Direct detection of fragments that bind to individual components of a complex or that perturb PPIs Kd Three-dimensional structural information	Bcl-XL primary screening and structural studies using ^Citation1^Citation5N/^Citation1H HSQC^Citation57 Mcl-1 primary screening using SOFAST ^Citation1H-^Citation1^Citation5N HMQC^Citation58 XIAP/clAP1 structural studies using ^Citation1^Citation5N/^Citation1H HSQC^Citation62-Citation64
Ligand observed       STD-NMR	Primary screening Hit validation	Detection of binding to individual components of a complex through crossrelaxation in the protein-fragment complex	BRCA2/RAD51 hit validation using STD-NMR^Citation67 Arfl-Arno hit validation using ^Citation1H STD-NMR^Citation77
Water-LOGSY	Primary screening Hit validation	Detection of binding to individual components of a complex through cross-relaxation in the fragment/protein-bound water molecules	XIAP/clAP1 primary screening using ID-NMR^Citation62-Citation64
X-ray crystallography	Primary screening Hit validation	Atomic resolution three-dimensional structure of fragment binding to individual components of a complex	Mcl-1 complex structure determination^Citation58 BRCA2/RAD51 complex structure determination^Citation67 XIAP/clAP1 primary screening and complex structure determination^Citation62-Citation64 HCV NS3 primary screening and complex structure determination^Citation20 Bromodomain primary screening^Citation73 and complex structure determination^Citation71-Citation74 Arf1-Arno complex structure determination^Citation77
SPR	Primary screening Competitive PPI assay Hit validation	Direct detection of fragments that bind to individual components of a complex or that interfere with PPIs Stoichiometry of binding Kd	Bromodomain Kd determination^Citation72 ArfI-Arno Kd determination^Citation77
ITC	Competitive PPI assay Hit validation	Direct detection of fragments that bind to individual components of a complex or that interfere with PPIs Stoichiometry of binding Kd Thermodynamic (enthalpic and entropic) binding contributions	BRCA2/RAD51 Kd determination^Citation67 XIAP/clAP1 Kd determination^Citation62-Citation64 HCV NS3 Kd determination^Citation20
TSA	Primary screening Hit validation	Detection of fragments that stabilize the temperature-dependent protein unfolding of individual components of a complex	BRCA2/RAD51 primary screening^Citation67 Bromodomain hit validation^Citation72
Biochemical assay	Primary screening	Direct detection of fragments that perturb PPIs	Bcl-XL/BH3 peptide FP competition assay^Citation57
FP	Competitive PPI assay		Mcl-I/BH3 peptide FP competition assay^Citation58
FRET	Hit validation	Ki for ligand displacement competition assays IC50 (if fragment binding modulates enzyme activity)	XIAP/clAPI IC50 determination^Citation62-Citation64 HCV NS3 IC50 determination using FRET-based assay^Citation20 Bromodomain primary screening using FP and FRET- based assays^Citation71–Citation72–Citation74 ArfI-Arno FP assay to triage virtual screening hits^Citation77

Notes: A typical screening cascade comprises two or more orthogonal techniques to identify binders and triage hits. “Primary screening” refers to high-throughput techniques that can be used to screen a fragment library and detect preliminary hits. “Competitive PPI assay” refers to secondary screening techniques that are capable of directly detecting fragments that interfere with a PPI. “Hit validation” refers to information-rich techniques that can be used to cross-validate primary hits and determine their characteristics – for example, affinity (NMR/SPR/ITC/biochemical assay), binding mode, or three-dimensional structure (NMR/X-ray crystallography).

Abbreviations: 2D-HMQC, two-dimensional heteronuclear multiple-quantum correlation; 2D-HSQC, two-dimensional heteronuclear single quantum correlation; FP, fluorescence polarization; FRET, fluorescence resonance energy transfer; HCV, hepatitis C virus; IC₅₀, half maximal inhibitory concentration; ITC, isothermal titration calorimetry; K_d, binding affinity; K_i, inhibition constant; NMR, nuclear magnetic resonance; PPI, protein–protein interaction; SOFAST, selective optimized flip angle short transient; SPR, surface plasmon resonance; STD-NMR, saturation transfer difference NMR; TSA, thermal shift assay; Water-LOGSY, Water-Ligand Observed via Gradient SpectroscopY.

Table 2 Initial fragment hits and current leads for protein–protein interaction case studies

Target	Example initial fragment hit	Example optimized structure
BCL-XL^Citation5,Citation57
Mcl-1^Citation58
XIAP/clAP1^Citation62-Citation64
HCV-NS3^Citation20
BRCA2-RAD51^Citation67
Bromodomains (i)^Citation71		N/A
Bromodomains (ii)^Citation72
Bromodomains (iii)^Citation74
Bromodomains (iv)^Citation75
Arfl-Arno ^Citation77		N/A

Abbreviations: HCV, hepatitis C virus; N/A, not applicable.

Toogood PL. Inhibition of protein–protein association by small molecules: approaches and progress. J Med Chem. 2002;45:1543–1558.

 PubMed Web of Science ®Google Scholar

Tse C, Shoemaker AR, Adickes J, et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 2008;68:3421–3428.

 PubMed Web of Science ®Google Scholar

Petros AM, Dinges J, Augeri DJ, et al. NMR-derived structure of the inhibitor N-(4″-fluorobiphenyl-4-ylcarbonyl)-3-nitro-4-(2-phenylsulfanylethyl amino)benzenesulfonamide bound to the antiapoptotic protein Bcl-xL. J Med Chem. 2006;49:656–663.

 PubMed Web of Science ®Google Scholar

Friberg A, Vigil D, Zhao B, et al. Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design. J Med Chem. 2013;56(1):15–30.

 PubMed Web of Science ®Google Scholar

Chessari G, Buck I, Coyle J, et al. Novel small molecule antagonists of XIAP, cIAP1/2 generated by fragment-based drug discovery. Poster presented at the ESH International Conference on Mechanisms of Cell Death and Disease: Advances in Therapeutic Intervention and Drug Development; October 14–18, 2010; Cascais, Portugal.

 Google Scholar

Ahn M, Ward G, Chessari G, et al. Potent, dual cIAP1/XIAP antagonists induce apoptosis in a melanoma stem cell population. Poster presented at AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics Conference; October 19–23, 2013; Boston, MA.

 Google Scholar

Scott DE, Ehebauer MT, Pukala T, et al. Using a fragment-based approach to target protein–protein interactions. ChemBioChem. 2013;14:332–342.

 PubMed Web of Science ®Google Scholar

Rouhana J, Hoh F, Estaran S, et al. Fragment-based identification of a locus in the Sec7 domain of Arno for the design of protein–protein interaction inhibitors. J Med Chem. 2013;56:8497–8511.

 PubMed Web of Science ®Google Scholar

Saalau-Bethell SM, Woodhead AJ, Chessari G, et al. Discovery of an allosteric mechanism for the regulation of HCV NS3 protein function. Nature Chem Biol. 2012;8:920–925.

 PubMed Web of Science ®Google Scholar

Gehling VS, Hewitt MC, Vaswani RG, et al. Discovery, design, and optimization of isoxazole azepine BET inhibitors. ACS Med Chem Lett. 2013;4:835–840.

 PubMed Web of Science ®Google Scholar

Chung CW, Dean AW, Woolven JM, Bamborough P. Fragment-based discovery of bromodomain inhibitors part 1: inhibitor binding modes and implications for lead discovery. J Med Chem. 2012;55(2):576–586.

 PubMed Web of Science ®Google Scholar

Fish PV, Filippakopoulos P, Bish G, et al. Identification of a chemical probe for bromo and extra C-terminal bromodomain inhibition through optimization of a fragment-derived hit. J Med Chem. 2012;55(22):9831–9837.

 PubMed Web of Science ®Google Scholar

Bamborough P, Diallo H, Goodacre JD, et al. Fragment-based discovery of bromodomain inhibitors part 2: optimization of phenylisoxazole sulfonamides. J Med Chem. 2012;55(2):587–596.

 PubMed Web of Science ®Google Scholar

Zhao L, Cao D, Chen T, et al. Fragment-based drug discovery of 2-thiazolidinones as inhibitors of the histone reader BRD4 bromodomain. J Med Chem. 2013;56(10):3833–3851.

 PubMed Web of Science ®Google Scholar