טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMenasher Avi
SubjectDesign, Synthesis and Evaluation of New Mechanism-Based
Inhibitors of KDO8P Synthase
DepartmentDepartment of Biotechnology
Supervisor Professor Timor Baasov
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Bacterial resistance to commercially available antibiotics is recognized as a major current problem in infectious disease. Although there may be creative ways to circumvent resistance, the most effective long-term solution to problems of resistance is the development of drugs that act on heretofore-unexploited antibacterial targets. Toward this end, our group has developed a long-term research program to explore the structure and function of 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase, a new target for such drugs. The enzyme KDO8P synthase catalyzes the synthesis of a unique sugar KDO that is an essential component of the outer membrane lipopolysaccharide of Gram-negative bacteria. While its activity is essential for the full viability of Gram-negative bacteria, the enzyme does not exist in mammalian cells. This makes KDO8P synthase an attractive target for the development of new synthetic antibiotics.


In our earlier studies, by directly monitoring the enzymatic reaction on a millisecond time scale, we provided the first direct evidence that the enzyme acts upon the acyclic hemiketal phosphate intermediate. In addition, we have synthesized the first bisubstrate inhibitor of the enzyme that combines the key features of two substrates of the enzyme into a single molecule and demonstrated that this compound acts as a potent inhibitor of the enzyme with a Ki value of 0.4 mM. In attempts to further improve the inhibitory potency of this inhibitor, in the current work we designed and synthesized its two different derivatives: the one is N-oxo derivative and the second is the homo-isosteric phosphonate. These new structures were then analyzed as inhibitors of the enzyme from two different origins: one not requires metal ions for the activity and the second that requires divalent metal ions for its full catalytic activity.  From the data obtained it turns out that these new analogs are the most potent inhibitors of the enzyme (of both classes) tested to date, with very significant improvement (by about two orders of magnitude) in inhibition of the metal-dependent enzyme. 


The primary importance of this research work is in rational design of new antibiotic drug that acts selectively on a new target in bacteria. This approach provides advantage relative to the modification of the existent antibiotics in terms of bacterial resistance. The observed strong inhibition of the new designed structures against the metal-dependant KDO8PS enzyme paves a way for further development of these compounds as novel antibiotics.