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.