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Screening Outside the Catalytic Site: Inhibition of Macromolecular Inter-actions Through Structure-Based Virtual Ligand Screening Experiments.

Sperandio O, Miteva MA, Segers K, Nicolaes GA, Villoutreix BO - Open Biochem J (2008)

Bottom Line: Protein-protein interactions play crucial roles in a number of biological processes, and, as such, their disruption or stabilization is becoming an area of intense activity.Along the same line, inhibition of protein-membrane could be of major importance in several disease indications.Despite the many challenges associated with the development of such classes of interaction modulators, there has been considerable success in the recent years.

View Article: PubMed Central - PubMed

Affiliation: Inserm U648, University of Paris 5, 45 rue des Sts Peres, 75006 Paris, France.

ABSTRACT
During these last 15 years, drug discovery strategies have essentially focused on identifying small molecules able to inhibit catalytic sites. However, other mechanisms could be targeted. Protein-protein interactions play crucial roles in a number of biological processes, and, as such, their disruption or stabilization is becoming an area of intense activity. Along the same line, inhibition of protein-membrane could be of major importance in several disease indications. Despite the many challenges associated with the development of such classes of interaction modulators, there has been considerable success in the recent years. Importantly, through the existence of protein hot-spots and the presence of druggable pockets at the macromolecular interfaces or in their vicinities, it has been possible to find small molecule effectors using a variety of screening techniques, including combined virtual ligand-in vitro screening strategy. Indeed such in silico-in vitro protocols emerge as the method of choice to facilitate our quest of novel drug-like compounds or of mechanistic probes aiming at facilitating the understanding of molecular reactions involved in the Health and Disease process. In this review, we comment recent successes of combined in silico-in vitro screening methods applied to modulating macromolecular interactions with a special emphasis on protein-membrane interactions.

No MeSH data available.


Related in: MedlinePlus

Example of one small molecule identified in the FV-membrane study that impedes protein-membrane interaction, it can be used as a mechanistic probe to investigate this molecular event or, could be used as a starting point to develop anticoagulant molecules.
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Figure 6: Example of one small molecule identified in the FV-membrane study that impedes protein-membrane interaction, it can be used as a mechanistic probe to investigate this molecular event or, could be used as a starting point to develop anticoagulant molecules.

Mentions: SB-VLS methods help to prioritize large compound collections prior to experimental testing. We applied our validated multi-step SB-VLS protocol [72] to both, the open and closed C2 crystal forms and selected the best 500 molecules for each receptor form (i.e., we decided to screen around 1000 compounds in total). This hierarchical screening protocol starts with using a relatively large compound collection (of about 500,000 molecules in 2D) obtained from a chemical vendor (in the present case it was ChemBridge [73]). Some molecules that do not appear to have a drug-like profile or that contain reactive groups are removed using several soft ADME/Tox filtering steps [74]. It is important to note that at this level of investigation, the filtering was relatively soft in order to keep a significant number of molecules for the subsequent phases of the procedure. After this ADME/Tox step, about 200,000 molecules were removed and the remaining compounds were transformed to a multiconformer collection in 3D. Each compound was docked as a rigid body and the molecules that had the best shape complementarity with the receptor were kept for an additional flexible docking step. This latter used an incremental construction algorithm as implemented in the Surflex package [75, 76]. Subsequently, the highest-ranking molecules in term of predicted affinity (scores) were selected for experimental assays. We screened experimentally 1018 molecules and we initially studied their ability to inhibit prothrombin activation. Nine molecules were identified that inhibited a prothrombinase assay by more than 99% at a concentration of 100 μM. Two molecules were inhibiting the assay system via a membrane-independent mechanism but seven molecules remained, apparently selectively inhibiting the FV-membrane interaction (see an example Fig. 6). To confirm that these compounds were impeding the association between of FV C2 domain with the membrane, we performed surface plasmon resonance (SPR) experiments. Results of these direct binding analyses confirmed that the small molecules were indeed inhibiting the protein-membrane interaction and we noticed that four out of the seven hit compounds also inhibited membrane binding of the structurally related blood coagulation FVIII.


Screening Outside the Catalytic Site: Inhibition of Macromolecular Inter-actions Through Structure-Based Virtual Ligand Screening Experiments.

Sperandio O, Miteva MA, Segers K, Nicolaes GA, Villoutreix BO - Open Biochem J (2008)

Example of one small molecule identified in the FV-membrane study that impedes protein-membrane interaction, it can be used as a mechanistic probe to investigate this molecular event or, could be used as a starting point to develop anticoagulant molecules.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570552&req=5

Figure 6: Example of one small molecule identified in the FV-membrane study that impedes protein-membrane interaction, it can be used as a mechanistic probe to investigate this molecular event or, could be used as a starting point to develop anticoagulant molecules.
Mentions: SB-VLS methods help to prioritize large compound collections prior to experimental testing. We applied our validated multi-step SB-VLS protocol [72] to both, the open and closed C2 crystal forms and selected the best 500 molecules for each receptor form (i.e., we decided to screen around 1000 compounds in total). This hierarchical screening protocol starts with using a relatively large compound collection (of about 500,000 molecules in 2D) obtained from a chemical vendor (in the present case it was ChemBridge [73]). Some molecules that do not appear to have a drug-like profile or that contain reactive groups are removed using several soft ADME/Tox filtering steps [74]. It is important to note that at this level of investigation, the filtering was relatively soft in order to keep a significant number of molecules for the subsequent phases of the procedure. After this ADME/Tox step, about 200,000 molecules were removed and the remaining compounds were transformed to a multiconformer collection in 3D. Each compound was docked as a rigid body and the molecules that had the best shape complementarity with the receptor were kept for an additional flexible docking step. This latter used an incremental construction algorithm as implemented in the Surflex package [75, 76]. Subsequently, the highest-ranking molecules in term of predicted affinity (scores) were selected for experimental assays. We screened experimentally 1018 molecules and we initially studied their ability to inhibit prothrombin activation. Nine molecules were identified that inhibited a prothrombinase assay by more than 99% at a concentration of 100 μM. Two molecules were inhibiting the assay system via a membrane-independent mechanism but seven molecules remained, apparently selectively inhibiting the FV-membrane interaction (see an example Fig. 6). To confirm that these compounds were impeding the association between of FV C2 domain with the membrane, we performed surface plasmon resonance (SPR) experiments. Results of these direct binding analyses confirmed that the small molecules were indeed inhibiting the protein-membrane interaction and we noticed that four out of the seven hit compounds also inhibited membrane binding of the structurally related blood coagulation FVIII.

Bottom Line: Protein-protein interactions play crucial roles in a number of biological processes, and, as such, their disruption or stabilization is becoming an area of intense activity.Along the same line, inhibition of protein-membrane could be of major importance in several disease indications.Despite the many challenges associated with the development of such classes of interaction modulators, there has been considerable success in the recent years.

View Article: PubMed Central - PubMed

Affiliation: Inserm U648, University of Paris 5, 45 rue des Sts Peres, 75006 Paris, France.

ABSTRACT
During these last 15 years, drug discovery strategies have essentially focused on identifying small molecules able to inhibit catalytic sites. However, other mechanisms could be targeted. Protein-protein interactions play crucial roles in a number of biological processes, and, as such, their disruption or stabilization is becoming an area of intense activity. Along the same line, inhibition of protein-membrane could be of major importance in several disease indications. Despite the many challenges associated with the development of such classes of interaction modulators, there has been considerable success in the recent years. Importantly, through the existence of protein hot-spots and the presence of druggable pockets at the macromolecular interfaces or in their vicinities, it has been possible to find small molecule effectors using a variety of screening techniques, including combined virtual ligand-in vitro screening strategy. Indeed such in silico-in vitro protocols emerge as the method of choice to facilitate our quest of novel drug-like compounds or of mechanistic probes aiming at facilitating the understanding of molecular reactions involved in the Health and Disease process. In this review, we comment recent successes of combined in silico-in vitro screening methods applied to modulating macromolecular interactions with a special emphasis on protein-membrane interactions.

No MeSH data available.


Related in: MedlinePlus