27 May 2015

Stopping Virulence...One Fragment at a Time.

The best way to not get an infectious disease is vaccinate.   Streptococcus pneumoniae is repsonsible for a million deaths world-wide every year.  For Streptococcus pneumoniae, there are a numbers of vaccines on the market.  These vaccines are bacterial polysaccharides either naked or conjugated to a protein.  They are highly effective, but don't cover all serotypes (there are ~100).  And sometimes a novel serotype arises.  So, if you do get infected treatment is key.  Beta-lactams are the first line of defense, but multi-drug resistance is on the rise, so alternate forms of treatment are needed. Targeting virulence factors has become a recent line of research.  Pneumococcal surface antigen A (PsaA) is strictly conserved surface-exposed lipoprotein expressed by all known pneumococcal serotypes and is essential for colonization and pathogenesis.  PsaA is an integral part of an ATP-binding cassette(ABC) transporter protein complex known as the PsaBCA permease, which is involved in manganese (Mn2+) transport across the bacterial cell membrane. (See there's always a metal involved in cool biology.)  This makes PsaA a good target for pneumococcal infections.  In this paper, a group from down under presents their results using fragments to target PsaA.

They custom built a fragment library (via outsourcing) ~1500 fragments.  This struck me as unusual, if not unique.  Typically, academics make their own or just buy one off the shelf.  I would love to hear why this path was chosen.  In the SI, they do say they used "relaxed" Ro3, but the only relaxation seems to be on the MW.  Have other academics gone this route?  I would love to know more (you can be anonymous in the comments, hint hint).  These were docked into the PsaA metal binding site (Figure 1) based on 3D shape and electrostatic similarity. These were then scored using FlexX. 
Figure 1.  Structure of PsaA. 
The top 300 fragments were manually inspected and then subjected to a cluster analysis.  The 60 most diverse fragments were then tested in a competitive Zn-binding assay.  Zn is a irreversible inhibitor of PsaA and the assay uses this to test for compound binding.  10 of the 60 fragments exhibited greater than 15% inhibition at 100 microM.  Two of these compounds showed greater than 50% inhibition at 1mM (Cpd 15 and 58, Figure 2.)
Figure 2.  Fragments with greater than 50% activity at 1 mM.  Hydrogen bond acceptors are shown in red, H-bond donors in Blue.
So, with crystal structures available, the authors decided to inspect the docked poses rather than actually trying to obtain a structure of the fragments bound to the protein. So even though docked fragments can, and do tend to, keep their original locations, experimental data is key to confirming in silico predictions.  The made 31 compounds around 15, and one that replaced the p-nitro, o-methoxy phenyl with o-hydroxypphenyl was the best 15h (28 microM, pIC50/HAC=0.37).  To that end, they tried to soak apo-crystals with cpd 15h and were unsuccessful due to limited compound solubility and affinity for the target.  They did not attempt soaking compound 58, which they was unable to be further "optimized" with simple SAR.  Cpd 15h did have antimicrobial activity: significant growth inhibition at 180 ug/ml and total growth inhibition at ~800 ug/ml.  They did a further round of optimization.

This is an example of real FBDD approach, in contrast to just using the words.  However, I think this is really a MPU (minimal publishable unit).  If we are lucky, we can expect to see future papers coming out describing their success (or failure) against this target. 


Dan Erlanson said...

The most active molecules are almost certainly metal chelators, so this is reminiscent of Seth Cohen's designed library of metallophilic fragments. As in that case, one challenge will be achieving suitable selectivity.

Joe Bauman said...

I'm confused by their virtual screening of a purchased library and then just testing the hits from the virtual screen. If you are only going to test 60 compounds why not start with a much bigger virtual screen. I'm guessing that there was a lot more to the story as far as how this library came to being.

Dr. Teddy Z said...

My guess is that they didn't want to screen the entire library (but why pay to build one that big anyway) and figured the virtual screening would be a good way to winnow them down. I agree, there must be more to the story.

Anonymous said...

If you screen 100 uM compound in a metal dependent assay containing 500 nM metal then its not surprising you find metal chelators. If you get really lucky, some of them might bind to protein too.

Peter Kenny said...

I’m on the road and so can’t actually see the article so it might be that there is a clear explanation for what caught my eye. There does seem to be a disconnect between one of the salicylanilide in the abstract and structure 15 in the blog post. From what I recall, the pKa of salicylanilide is around 6 (check because I’ve not looked at this for a very long time) and compounds that can deprotonate to give delocalized anions can sometimes function as oxidative phosphorylase uncouplers.

Compound 15 is unlikely to be significantly deprotonated under normal physiological conditions and I’d also anticipate a hydrogen bond between the amide NH and methoxy oxygen. The primary aryl sulfonamides is a pharmacophore that is associated with carbonic anhydrase inhibition and it is my understanding that the sulfonamide nitrogen deprotonates in order to bind the catalytic zinc cation. I recall primary aryl sulfonamides having a pKa values of about 10 so increasing acidity would be an obvious design tactic (for carbonic anhydrase at least). From what I recall, the compound with a primary sulfonamide at C2 of benzothiazole has a pKa just below 8 and this compound appears to be commercially available.