Caren L. Freel Meyers

The continued widespread exposure of human pathogens to anti-infective agents fosters the inevitable evolution of resistance mechanisms in clinically relevant pathogens, and the emergence of antibiotic resistance in human pathogens that cause life-threatening infections has occurred at an alarming rate in almost every major class of anti-infective agents. The fight against rapid progression of clinical resistance to anti-infective agents demands the sustained discovery and development of new agents and exploration of novel anti-infective targets. Our long-term goal is to develop novel approaches to kill human pathogens, including bacterial pathogens and malaria parasites, with the ultimate objective of developing potential therapeutic agents. Toward this goal, we are pursuing studies of bacterial isoprenoid biosynthetic enzymes comprising the methylerythritol phosphate (MEP) pathway essential in many human pathogens (Figure 1). Studies focus on understanding mechanism throughout the pathway toward the development of selective inhibitors of isoprenoid biosynthesis. Our strategies for creating new anti-infective agents involve interdisciplinary research in the continuum of organic, biological and medicinal chemistry. Molecular biology, protein expression and biochemistry, and synthetic chemistry are key tools for our research.

Mechanistic studies and selective inhibition of DXP synthase: The first step in the MEP pathway is catalyzed by thiamin-diphosphate (ThDP)-dependent DXP synthase. The product, DXP, is required for production of essential bioprecursors, IPP and DMAPP, in pathogen isoprenoid biosynthesis and also serves as a precursor in vitamin B1 and vitamin B6 biosynthesis. We are pursuing selective inhibitors of DXP synthase toward the development of new anti-infective agents. Mechanistic studies from our lab suggest that this enzyme utilizes a unique rapid equilibrium, random sequential mechanism (Figure 2). The requirement of a ternary complex in DXP synthase catalysis leads to the idea that this enzyme can be selectively targeted by inhibitors that occupy a large active site that uniquely accommodates both substrates. This knowledge combined with the observation that DXP synthase shows flexibility toward non-polar acceptor substrates has led to the design and synthesis of unnatural bisubstrate analogs which exhibit selective inhibition against DXP synthase (Figure 3). Butylacetylphosphonate (BAP) exhibits considerably more potent inhibitory activity against DXP synthase compared to ThDP-dependent enzymes pyruvate dehydrogenase (PDH) and transketolase (TK). These studies serve as an excellent starting point for the design of more potent, selective inhibitors of this essential enzyme.    

Mechanistic studies of IspG: The sixth enzyme in the MEP pathway is catalyzed by iron-sulfur enzyme IspG. Reductive ring opening of MecPP leads to the formation of HMBPP (Figure 1). We have synthesized a linear epoxide and determined, through collaboration with the Liu lab (Boston University),­ that the epoxide serves as an excellent substrate for IspG and is also capable of undergoing epoxide opening to re-form MEcPP, supporting the idea of epoxide formation during catalysis by IspG. Current studies build on this observation to selectively target IspG.

DRUG DELIVERY

A prodrug is a pharmacologically inactive compound that is converted to an active drug via a biological activation process that ideally takes place at the site of action. Several reasons exist for the utilization of prodrug strategies in drug design including improvement of solubility, absorption and distribution, site specificity, metabolic or chemical instability of the parent drug, prolonged release, toxicity, poor patient acceptability and problems with formulation.

Intracellular delivery of diphosphate analogs:  Current efforts in our lab focus on intracellular delivery of polyphosphorylated molecules, including clinically-used bisphosphonates, for the treatment of cancer and/or infectious diseases. Bisphosphonates are used for the treatment of a variety of bone disorders. The polyanionic nature of these compounds, which promotes rapid localization to the bone matrix, prevents efficient cellular uptake in soft tissues and therefore severely limits their use for the treatment of extraskeletal diseases. We have developed a bisphosphonamidate prodrug strategy for the intracellular delivery of bisphosphonates that relies upon minimal enzymatic activation events to release multiple negative charges. Bisphosphonamidates exhibit potent anticancer activity and a remarkable enhancement in potency compared to the parent bisphosphonates (Figure 4).

Anti-infective prodrugs:  Despite the success of prodrug strategies with cancer treatment, few examples are documented of the use of prodrug approaches for the treatment of bacterial infections. We are pursuing the development of antibiotic prodrug approaches for the delivery of drugs that exhibit potent antibiotic activity but exhibit problems of low solubility, poor pharmacokinetics and toxicity. Our laboratory is designing antibiotic prodrugs that will undergo activation to liberate multiple drug molecules aimed at multiple bacterial targets simultaneously within a single bacterial cell.