LITERATURE REVIEW: Moving the Druggable Needle

Efforts to cover novel chemical space for drug discovery (e.g., outside of typical sp2-rich compounds) have included natural products, “3D” chiral structures such as found in Chiromics libraries, macrocyclic libraries, and the use of DNA-encoded libraries, which enable screening of libraries containing ∼100 million members. This paper describes the use of an mRNA display technology and affinity-based selection to cover a macrocyclic peptide library to find inhibitors of phosphoglycerate mutases (PGM). This enzyme operates in glycolysis and gluconeogenesis to catalyze the interconversion of 2- and 3-phosphoglycerate (2PG, 3PG). Inhibitors of PGM are of high interest to treat parasitic diseases such as those caused by nematode worms because PGM is an essential enzyme for nematode survival, and the nematode PGM enzyme (iPGM) is unrelated both in sequence and catalytic mechanism to mammalian PGM enzyme (dPGM).

Previous efforts to find inhibitors of this enzyme using typical low-molecular compound libraries have not been successful. The mRNA display technology employed in this work is known as RaPID (random nonstandard peptides integrated discovery) and enabled paneling trillions of cyclic peptides to identify the small fraction that bind with high affinity to iPGM. The peptides all contain an L- or D-N-chloroacetyl tyrosine at the N-terminus and 19 additional amino acids including a cysteine. Macrocyclization of the peptide occurs through nucleophilic attack of the cysteine thiolate on the chloroacetyl group. Incorporation of puromycin in the mRNA covalently links the peptide to the mRNA at the peptide’s C-terminus.

Following several rounds of affinity selection against bead-bound iPGM, the enriched peptide mRNAs are sequenced and the peptides can then be chemically synthesized to confirm activity. Initial paneling of iPGM from the parasite Brugia malayi against a D-tryosine library yielded macrocycles of modest potency (single-digit μM). The B. malayi iPGM enzyme also proved difficult to crystalize for structural analysis, so subsequent efforts focused on iPGM from Caenorhabditis elegans which readily formed high quality crystals. However, affinity selection against C. elegans iPGM with both D and L-tryrosine libraries led to the identification of several high affinity macrocyclic peptides (so-called ipglycermides). Ipglycermide B (Ce-2; Figure) was determined to bind with a KD = 73 ± 15 pM. Stoichiometric binding was demonstrated by varying the iPGM concentration in the enzyme assay from nM to pM concentrations (Figure).

Examination of linear analogues supports that a good deal of the potency is derived from macrocyclization of the peptide, which reduces the entropic cost of binding. Co-crystal structures of a truncated analog (Ce-2d; Figure) revealed that this ligand binds to an allosteric site between the transferase and phosphatase domains, which is consistent with the potency for the ipglycermides being independent of 3PG concentration. Ipglycermide B shows pan-orthologue potency against four parasitic iPGMs as well as Escherichia coli iPGM but no inhibitory activity at Homo sapiens dPGM or E. coli dPGM. The co-crystal structures suggested that a cysteine present at position 14 of ipglycermide B is in position to ligate the catalytic zinc present in iPGM. Substitution of serine at this position or truncation of the peptide (Ce-2d, ipglycermide B4; Figure) results in a loss in affinity of approximately one log against C. elegans iPGM and improved orthologue selectivity against other parasitic or E. coli iPGMs while maintaining inactivity against human dPGM. In addition, the orthologue selectivity of Ce-2d follows the phylogenetic relationship of enzymes (Figure). This paper demonstrates how the application of new chemical matter on a massive scale along with an efficient selection strategy can be used to find potent ligands for difficult targets, serving as starting points for drug development.

Following several rounds of affinity selection against bead-bound iPGM, the enriched peptide mRNAs are sequenced and the peptides can then be chemically synthesized to confirm activity. Initial paneling of iPGM from the parasite Brugia malayi against a D-tryosine library yielded macrocycles of modest potency (single-digit μM). The B. malayi iPGM enzyme also proved difficult to crystalize for structural analysis, so subsequent efforts focused on iPGM from Caenorhabditis elegans which readily formed high quality crystals. However, affinity selection against C. elegans iPGM with both D and L-tryrosine libraries led to the identification of several high affinity macrocyclic peptides (so-called ipglycermides). Ipglycermide B (Ce-2; Figure) was determined to bind with a KD = 73 ± 15 pM. Stoichiometric binding was demonstrated by varying the iPGM concentration in the enzyme assay from nM to pM concentrations (Figure).

Examination of linear analogues supports that a good deal of the potency is derived from macrocyclization of the peptide, which reduces the entropic cost of binding. Co-crystal structures of a truncated analog (Ce-2d; Figure) revealed that this ligand binds to an allosteric site between the transferase and phosphatase domains, which is consistent with the potency for the ipglycermides being independent of 3PG concentration. Ipglycermide B shows pan-orthologue potency against four parasitic iPGMs as well as Escherichia coli iPGM but no inhibitory activity at Homo sapiens dPGM or E. coli dPGM. The co-crystal structures suggested that a cysteine present at position 14 of ipglycermide B is in position to ligate the catalytic zinc present in iPGM. Substitution of serine at this position or truncation of the peptide (Ce-2d, ipglycermide B4; Figure) results in a loss in affinity of approximately one log against C. elegans iPGM and improved orthologue selectivity against other parasitic or E. coli iPGMs while maintaining inactivity against human dPGM. In addition, the orthologue selectivity of Ce-2d follows the phylogenetic relationship of enzymes (Figure). This paper demonstrates how the application of new chemical matter on a massive scale along with an efficient selection strategy can be used to find potent ligands for difficult targets, serving as starting points for drug development.

* Abstract from Nat Commun 2017;8:14932

Glycolytic interconversion of phosphoglycerate isomers is catalysed in numerous pathogenic microorganisms by a cofactor-independent mutase (iPGM) structurally distinct from the mammalian cofactor-dependent (dPGM) isozyme. The iPGM active site dynamically assembles through substrate-triggered movement of phosphatase and transferase domains creating a solvent inaccessible cavity. Here we identify alternate ligand binding regions using nematode iPGM to select and enrich lariat-like ligands from an mRNA-display macrocyclic peptide library containing >1012 members. Functional analysis of the ligands, named ipglycermides, demonstrates sub-nanomolar inhibition of iPGM with complete selectivity over dPGM. The crystal structure of an iPGM macrocyclic peptide complex illuminated an allosteric, locked-open inhibition mechanism placing the cyclic peptide at the bi-domain interface. This binding mode aligns the pendant lariat cysteine thiolate for coordination with the iPGM transition metal ion cluster. The extended charged, hydrophilic binding surface interaction rationalizes the persistent challenges these enzymes have presented to small molecule screening efforts highlighting the important roles of macrocyclic peptides in expanding chemical diversity for ligand discovery.