Although, it has been confirmed that the formation of homodimers of PEX9 is what leads to biological activity [ 67 , 68 ]. This was confirmed by electrophoresis, atomic force microscopy and transmission electron microscopy [ 69 ]. It was remarked that the pro-MMP9 trimer demonstrated a higher affinity towards TIMP-1 than its monomer with a fold difference in magnitude. Cha et al. A disulfide bond between Cys and Cys connected them to maintain the integrity of the structure. A highly conserved disulfide bond was present amongst all previous PEX domains as shown in both monomeric and dimeric states.
It is to be noted that the homodimerisation of PEX9 is reduction-sensitive, which was understood at that time to mean that dimerisation was linked by disulfide bonds. However, later investigations showed that the link between the monomers did not belong to the disulfide bond variety, but rather a hydrophobic interaction between blades 4 for each sub-unit.
This disulfide bond is easily cleaved by reducing agents, leading to dimer dissociation and explaining the sensitivity of the pro-MMP9 dimer. The crystallised PEX9 structure has no inhibitor bound within any pocket; however, there are two sulfate ions bound at blades 1 and 3. The sulfate was bound to this site with a strong interaction to the Arg residue measured to be 3. Considering that there are no other PEX9 holoproteins, the binding site of this sulfate ion could be the starting point to predict inhibitor-PEX9 interactions using a structure-based computational design.
These 2 domains are connected by 17 amino acids which might be flexible and free of secondary structure. They showed an additional sodium, calcium and chloride ion bound to the protein by forming a channel-like opening PDB 1GEN [ 72 ]. When PEX13 was crystallised, it also revealed a similar pseudo-fourfold symmetry assigning 2 calcium, 2 chloride and additional sulfate ions in the central tunnel tube [ 74 ]. The 3D structure of hemopexin domains. This domain also shows homodimerisation in which blade 2 and blade 3 of chain A interact hydrophobically with blade 3 and 2 of chain B.
The interruption of this homodimerisation such as amino acid mutagenesis affects the functionality of MT1-MMP in pro-MMP2 activation as well as collagen degradation. In contrast, the non-dependent dimer functions including gelatin film degradation and cell migration are not affected [ 75 ]. Because it is known that MMPs are involved in many pathological processes, it was a matter of time before the first MMP inhibitors were developed. However, this compound was found to be toxic during clinical phase [ 78 ]. The molecular structures of batimastat and marimastat see Fig.
Thus, they act as reversible competitive inhibitors towards the MMPs [ 5 , 79 , 80 ]. The next generation of MMP inhibitors was still designed to target the metalloenzyme at the catalytic site [ 81 , 82 ]; therefore, the coming issue would be either drug resistance due to the non-selective binding mode of inhibitors towards the catalytic site or the adverse side effects such as musculoskeletal defects. The hemopexin domain in MMP9 has a different structure compared to the other MMPs, which means that this domain could be the next interesting target in the discovery of MMP9-selective inhibitors to treat cancer diseases [ 83 ].
It underwent a clinical phase 2 study in the treatment of chronic obstructive pulmonary disease COPD. However, more studies are necessary to be carried out for further developments [ 85 ]. The structures of some MMP inhibitors are presented in Fig. Kalva et al. The crystal structure being used was 1GKC, a complex of MMP9 with an inhibitor bearing a hydroxamate functional group.
The structure-based pharmacophore modelling was incorporated with molecular docking studies, which revealed two aromatic rings and three hydrogen bond acceptors as the features of an MMP9 inhibitor, although this is for the catalytic domain and not for binding to PEX9. The pharmacophore model generated by Kalva was re-drawn in Fig.
The pharmacophore model of hinokiflavone is composed of two aromatic rings brown and hydrogen bond acceptors cyan at the measured distances in angstroms. Uniquely, the peptide generated could not compete with TIMP-1 as the natural inhibitor; therefore, the peptide does not directly affect the proteolytic activity of MMP9.
Furthermore, the prevention of pro-MMP9 activation followed by blocking of cell migration as well as in vivo tumour cell growth indicates the selectivity of PEX9 to be used as the target [ 87 ]. Drug modelling aided by computers has been commonly used to rationalise the constructed model before a drug is chemically synthesised and biologically tested [ 88 ].
The advances of computer technology, complemented with robust data from bioinformatics and chemo-informatics, are extremely helpful in doing tasks involving drug design and discovery in respect to reducing the cost and increasing the speed of the investigation [ 89 ].
There are several software available that facilitate structure-based drug design when the crystal structure of the protein is available [ 90 ] and ligand-based drug design when only the ligand with its biological activity is available [ 91 ]. The software could be free, e. The bioinformatics data such as the protein crystal structure, either in apo-form or holo-form, is freely downloadable from protein databases such as the Protein Data Bank, commonly abbreviated as PDB accessible at www.
Alternatively, if the ligand is a new chemical entity, its molecular structure could be directly drawn based on their chemical structures shown in published articles. The utilisation of a computer-aided drug modelling is able to shorten the time of investigations because the software can screen millions of ligands to be shortlisted as hit compounds by in silico testing. Two studies utilised this crystal structure as the model in a virtual screening of the ZINC database in the effort to discover MMP9 inhibitors which can selectively bind to the PEX domain, as explained below.
Dufour et al. A hundred ligands had been selected from ZINC and five top in silico hits 6 — 10 which can be seen in Fig. This was used to investigate their capabilities in inhibiting the activity of the PEX9 domain. The binding of five compounds towards PEX9 was observed upon fluorescence assay, revealing the dissociation constant K d for each compound associated with their binding affinities. The compounds were then checked for their cytotoxicities against COS-1 cells to measure the LD 50 , and it was followed by cell proliferation determination upon treatment with the compounds.
Finally, the in vivo experiment was carried out to evaluate the MMP9 expression interference in mice using a tumour xenograft model, i. An early conclusion was taken that only compounds 6 and 7 did not cause a notable cytotoxicity in COS-1 cells; therefore, both compounds blocking the MMP9-induced cell migration were not because of their toxicities but in respect to their selectivity towards cancer cells.
The binding of compound 7 with PEX9 resulted in a K d of 2. Structurally, compound 7 has a six-member heterocycle ring which provides a planar conformation that fits into the cavity of PEX9 domain blades, whereas the arylamide moiety facilitates a more flexible conformation, which bound to the surface near the cavity.
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Therefore, this experiment highly correlates with the in vitro study that compound 7 successfully disrupts the MMP9-induced cell migration via binding with the PEX domain as an allosteric inhibitor. The molecular docking was carried out with a protocol similar to the one that was used by Dufour. There were two binding sites used by Li wherein the sulfate ion was bound to the interface of four blades site 1 and at the interface of blade 3 and blade 4 site 2. The binding assay study revealed the K d of four hit compounds 11 , 12 , 13 and 14 in low micromolar inhibitions 0.
The structure of those four compounds 11 — 14 are presented in Fig. From the results, it is most likely that the steroid backbone is still dominant to construct the ligand-protein binding. The four hit compounds active against PEX9 selected from NCI database 11—14 [ 98 ], the derivatives of 7 15 [ 98 ] and 16 and the docking pose of 16 at PEX9 binding site in a protein surface form 16a and stick form 16b. The latest finding on small molecule inhibitors targeting the PEX9 domain of latent MMP9 was carried out by [ ], demonstrating a prevention of focal adhesion junction formation associating with specific protein-ligand binding.
Computational docking is used to design the compound series which tightly dock to the PEX9 binding site. One of the interesting compounds 15 generated from 7 bears a flexible n -propyl chain within quinazolinone as the heterocyclic planar ring and p -fluoroarylamide ring. This compound demonstrated K d 0.
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The phosphorylation of SrC and its downstream is then decreased leading to the blocking of tumour cell growth, migration and invasion. According to the docking results, the fluorine atom was found deeply occupying within the binding pocket which is different from 7. This 7 shows solvent exposure surrounds difluoromethoxyphenol which decreases the free energy of binding. In addition, the planar quinazolinone was said to improve the ligand-binding efficiency as well as the longer flexible propyl chain.
These may contribute to the higher affinity of 15 than others.
The structure of 15 is illustrated in Fig. It was suggested that the ligand intervention at this centre point could defect the flexibility of homodimerisation, thus affecting the MMP14 activity. However, the hit compounds dominantly bear the sulfonamide moiety which is quite similar with most of the active ligands working on the catalytic site. Therefore, further study in the selectivity of compounds, which bind to either the catalytic or hemopexin site, should be carefully managed. Research in novel drug discovery for breast cancer by targeting the hemopexin domain of MMPs in general, and MMP9 specifically, is still less conducted.
To the best of our knowledge, there are only three publications reporting compounds inhibiting PEX9 including pyrimidine-arylamide [ , ] and steroid [ ] scaffolds.
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This is in contrast with studies targeting MMP9 with the catalytic domain as the protein target covering diverse scaffolds such as pyridinone, pyridithiol, biaryl ether sulfonamide hydroxamate, aryl sulfone, pyrimidine, aromatic carboxylic acid and flavonoid which have been reviewed in our previous published article [ 13 ]. The distinction of structure amongst the hemopexin domains is promising a better target for selective inhibition rather than the catalytic domains, which share highly conserved amino acid residues in their binding sites.
A potential compound from the ZINC database bearing a pyrimidinone scaffold extended by an arylamide moiety connected with a flexible S-alkyl chain was used to identify the pharmacophore which meets the requirements to be active as a PEX9 inhibitor.
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The pyrimidine ring has a planar character, which fits into the deep and stable hydrophobic pocket whereas the arylamide was said to be more flexible in the surface near to the cavity. Due to the unavailability of PEX9 in complex with its inhibitor, it is still not confirmed whether the binding site was correctly predicted by computational docking.
In the case where the protein crystal structure is not in complex with its inhibitor, homology modelling could be another strategy to conduct a structure-based drug design. This could be done if only there is another protein crystal structure in complex with inhibitor having high similarity in amino acid sequence with the desired protein structure being available.
This homologues crystal could be used as the template to model the desired 3D protein structure with the known binding site.
Intriguingly, two functional groups present in the compound that was identified by Dufour as being active against PEX9 compound 7 had not been addressed. A propyl group attached to the pyrimidine ring and the OCF 2 group at the para position of the arylamide were not assigned pharmacophores, which can be pointed out for further design. For example, by using compound 7 by Dufour, we can maintain the pivotal functional groups the planar ring and flexible arylamide followed by modifying the smaller functional groups to test bioisosterism.
The propyl group could be modified by extending the normal chain or cyclic chain, whereas the OCF 2 could be modified by substituting F with another halogen. The series of those modifications with their respective bioactivities could be processed as a QSAR model explaining the correlation whether it gives the compound a higher or lower bioactivity and what type of pharmacophore contributes to that correlation.
The pharmacophores that are marked as contributing to a negative value should be reduced or removed, whereas the positive correlations should be maintained or even added with the extra ones. Therefore, the modified functional group can keep being stable and have the correct conformation associated with their binding. Finally, we can conclude that from the data available, PEX9 is the selective target for MMP9 inhibition, which appears to have a significant advantage compared to other targets, leading to a potentially successful discovery in novel anti-breast cancer agents.
Our in-progress research is also generating a series of compounds bearing the pharmacophore as identified by Dufour et al. Here, we utilised purine ring to have heterocyclic planar character instead of pyrimidine has been used in the lead compounds. One of the compounds in the series is designed by attaching a strong electron withdrawing group such as NO 2 at the para-position of the arylamide ring This binding mode agrees with the study by Li  has been discussed previously. Interestingly, the in vitro inhibition assay of this compound towards MMP9 using fluorescence spectroscopy demonstrates IC 50 3.
The structures of 16 , 16a and 16b are illustrated in Fig. Hemopexin MMP9 is an interesting domain of MMP9 to be targeted in searching for more selective drug for breast cancer. The utilisation of computational drug design is able to develop more effective MMP9 inhibitor with a lower cost which could be applied for further lead optimisation using either structure-based drug design or ligand-based drug design.
This is a review article; therefore, data sharing is not applicable to this article as no datasets were generated or analysed during the current study. CA: Cancer J Clin — Int J Cancer E—E CA: Cancer J. Clin — Ministry of Health Republic of Indonesia: Situation of cancer disease.
J Cancer Sci Ther — FJPS; available online. Bauvois B New facets of matrix metalloproteinases MMP-2 and MMP-9 as cell surface transducers: outside-in signaling and relationship to tumor progression. BBA — Rev Cancer — Yang W, Li G The matrix metalloproteinases and cerebral ischemia. In: Balestrino M ed Advances in the preclinical study of ischemic stroke. Intech, London, pp — Aiken A, Khokha R Unraveling metalloproteinase function in skeletal biology and disease using genetically altered mice.
Wound Med — Cardiovasc Res — Oncotarget — Fields GB New strategies for targeting matrix metalloproteinases. Matrix Biol 44—— Physiology — Strongin AY Proteolytic and non-proteolytic roles of membrane type-1 matrix metalloproteinase in malignancy. Visse R, Nagase H Matrix metalloproteinases and tissue inhibitors of metalloproteinases.
Struct Funct Biochem — J Biol Chem — Clin Exp Metastasis — Cancer Treat Res Commun — BMC Cancer — Dis Markers — Breast Cancer Res Treat — J Med Biol Sci Res — McGowan PM, Duffy MJ Matrix metalloproteinase expression and outcome in patients with breast cancer: analysis of a published database.
Annu Oncol — Pathol Res Pract — Invest Ophthalmol Vis Sci — Sa-nguanraksa D, O-charoenrat P The role of vascular endothelial growth factor A polymorphisms in breast cancer. Int J Mol Sci — J Carcinog Neoplasia — Am J Cancer Res — Deryugina EI, Quigley JP Pleiotropic roles of matrix metalloproteinases in tumor angiogenesis: contrasting, overlapping and compensatory functions.
FEBS J — Lawler J Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med — Endocr Relat Cancer — Int J Oncol — Krstic J, Santibanez JF Transforming growth factor-beta and matrix metalloproteinases: functional interactions in tumor stroma-infiltrating myeloid cells. ScientificWorldJournal Each week you will learn the steps that a pharmaceutical or biotech company goes through to discover a new therapeutic drug.
We would highly recommend that you take the courses in order since it will give you a better understanding on how a drug is discovered in the lab before being tested in clinical trials and then launched in the market place. The course presents the basic concepts of DD and allows familiarization with the technical terms of the field. I strongly recommend for those who want to understand a little bit of DD. A very helpful and enlightening course.
Clears my basics of Drug Discovery and explains principles and aspects to be kept in mind when researching for a new drug.http://getlancer1.dev3.develag.com/the-pains-of-wandering-translated-by-mohammed-abu.php
Protein Discovery Technologies Drug Discovery Series 2009
In this module we will hear from Stephen Burley, M. Drug Discovery. Enroll for Free.