Investigating the Roles of Active Site Residues in Mycobacterium tuberculosis Indole-3-glycerol Phosphate Synthase, a Potential Target for Antitubercular Agents.

Mycobacterium tuberculosis drug resistance is emerging and new drug targets are needed. Tryptophan biosynthesis is necessary for M. tuberculosis replication and virulence. Indole-3-glycerol phosphate synthase (IGPS) catalyzes a step in M. tuberculosis tryptophan biosynthesis and has been suggested as a potential anti-infective target, but our understanding of this enzyme is limited. To aid in inhibitor design and gain a greater mechanistic picture of this enzyme, there is a need to understand the roles of active site amino acids in ligand binding and catalysis. In this work, we explored the roles of conserved active site amino acids Glu57, Lys59, Lys119, Glu168, and Glu219. Mutation of each to Ala results in loss of all detectable activity. The Glu57Gln, Lys59Arg, Lys119Arg, Glu168Gln, and Glu219Asp mutations result in large activity losses, while Glu219Gln has enhanced activity. Analysis of the enzymatic data yields the following main conclusions: (A) Lys119 is the likely catalytic acid in the CdRP ring closure step. (B) Glu168 stabilizes a charged reaction intermediate and may also be the catalytic base. (C) Glu57, Glu219, and Lys119 form a closely arranged triad in which Glu57 and Glu219 modulate the pKa of Lys119, and thus overall activity. This increased understanding of inter- and intramolecular interactions and demonstration of the highly coordinated nature of the M. tuberculosis IGPS active site provide new mechanistic information and guidance for future work with this potential new drug target.

Discovery of In Vivo Active Sphingosine-1-phosphate Transporter (Spns2) Inhibitors.

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that interacts with five G-protein-coupled receptors (S1P1-5) to regulate cellular signaling pathways. S1P export is facilitated by Mfsd2b and spinster homologue 2 (Spns2). While mouse genetic studies suggest that Spns2 functions to maintain lymph S1P, Spns2 inhibitors are necessary to understand its biology and to learn whether Spns2 is a viable drug target. Herein, we report a structure-activity relationship study that identified the first Spns2 inhibitor 16d (SLF1081851). In vitro studies in HeLa cells demonstrated that 16d inhibited S1P release with an IC50 of 1.93 µM. Administration of 16d to mice and rats drove significant decreases in circulating lymphocyte counts and plasma S1P concentrations, recapitulating the phenotype observed in mice made deficient in Spns2. Thus, 16d has the potential for development and use as a probe to investigate Spns2 biology and to determine the potential of Spns2 as a drug target.

Molecular recognition of HIV-1 RNAs with branched peptides.

Targeting RNA offers the potential in many diseases of a therapeutic treatment. Due to its large surface area and ability to adopt different conformations, targeting RNA has proven challenging. Medium-sized branched peptides are of the size to competitively bind RNA while remaining cell permeable, stable in vivo, and non-toxic. Additionally, the ease in generating a large library followed by high-throughput screening provides a way to suggest a scaffold with high diversity that is capable of targeting the structure and sequence of RNA. The ability to select various types of amino acid modifications in the branched peptide allows for variable structures and interactions of the branched peptide but can result in too large a task if not approached properly. In this chapter, we discuss a strategy to selectively recognize RNAs of interest through high throughput screening of branched peptides, validation of hits and biophysical characterization, leading by example with our experience in targeting HIV-1 RNAs with branched peptides.

Molecular recognition of a branched peptide with HIV-1 Rev Response Element (RRE) RNA.

Interaction of HIV-1 rev response element (RRE) RNA with its cognate protein, Rev, is critical for HIV-1 replication. Understanding the mode of interaction between RRE RNA and ligands at the binding site can facilitate RNA molecular recognition as well as provide a strategy for developing anti-HIV therapeutics. Our approach utilizes branched peptides as a scaffold for multivalent binding to RRE IIB (high affinity rev binding site) with incorporation of unnatural amino acids to increase affinity via non-canonical interactions with the RNA. Previous high throughput screening of a 46,656-member library revealed several hits that bound RRE IIB RNA in the sub-micromolar range. In particular, the lead compound, 4B3, displayed a Kd value of 410 nM and demonstrated selectivity towards RRE. A ribonuclease protection assay revealed that 4B3 binds to the stem-loop structure of RRE IIB RNA, which was confirmed by SHAPE analysis with 234 nt long NL4-3 RRE RNA. Our studies further indicated interaction of 4B3 with both primary and secondary Rev binding sites.

Discovery of a Branched Peptide That Recognizes the Rev Response Element (RRE) RNA and Blocks HIV-1 Replication.

We synthesized and screened a unique 46 656-member library composed of unnatural amino acids that revealed several hits against RRE IIB RNA. Among the hit peptides identified, peptide 4A5 was found to be selective against competitor RNAs and inhibited HIV-1 Rev-RRE RNA interaction in cell culture in a p24 ELISA assay. Biophysical characterization in a ribonuclease protection assay suggested that 4A5 bound to the stem-loop region in RRE IIB while SHAPE MaP probing with 234 nt RRE RNA indicated additional interaction with secondary Rev binding sites. Taken together, our investigation suggests that HIV replication is inhibited by 4A5 blocking binding of Rev and subsequent multimerization.