Synthesis of Peptidic, Natural Product-inspired, and Heterocyclic Molecules as Biological Probes

The first section of this thesis describes the solid phase peptide synthesis of scotophobin, a small peptide thought to be responsible for the transference of a learned response between mammals, and several related peptides. The synthetic peptides were tested against an array of G protein-coupled receptors. Although interesting activity was observed, these studies failed to provide closure to the storied past of scotophobin. We were able to demonstrate that the small peptide possesses in vitro activity. The second section describes the optimization of the thiol-mediated epoxide opening and intramolecular aldol reaction of epoxyketones. This methodology provided access to a variety of densely functionalized bicyclo[3.3.1]non-3-en-2-ones in moderate to good yield (64-88%). The newly synthesized bicyclo[3.3.1]non-3-en-2-ones were shown by a ChemGPS-NP analysis to occupy novel regions of chemical space. In addition, they exhibited moderate activity in several assays. The third section describes our efforts toward the total synthesis of chrysophaentin A. Although the synthesis of the natural product has yet to be achieved, the convergent synthesis of the monomeric C1-C16 tetraphenol of chrysophaentin A was completed in 10 steps (longest linear sequence) and 24% overall yield. The collaborative biological evaluation of two monomeric chrysophaentin A fragments revealed that they retained the potent antimicrobial activity of the parent natural product. The final section of this thesis describes the synthesis of three peptide-like inhibitors as well as several non-peptidic small-molecule inhibitors of botulinum neurotoxin serotype A light chain (BoNT/A LC). In collaborative work, all three peptidic inhibitors were found to possess sub-µM activity. X-ray crystallography was used to document the binding mode of one of the peptidic inhibitors on BoNT/A LC

Selecting an Anti-Malarial Clinical Candidate from Two Potent Dihydroisoquinolones

BACKGROUND: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described. METHODS: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs. RESULTS: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg. CONCLUSION: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators

Discovery of Novel, Orally Bioavailable, Antileishmanial Compounds Using Phenotypic Screening

Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 μM. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug

Iterative Structure-Based Peptide-Like Inhibitor Design against the Botulinum Neurotoxin Serotype A

The botulinum neurotoxin serotype A light chain (BoNT/A LC) protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs) were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their Ki values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2′ position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors

Discovery of an Orally Bioavailable Inhibitor of Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously reported the discovery, validation, and structure–activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1–UBE2M interaction. We demonstrated that compound <b>7</b> inhibits both the DCN1–UBE2M protein–protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound <b>7</b> exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CL_int = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (<b>67</b>). Compound <b>67</b> retains the favorable biochemical and cellular activity of compound <b>7</b> but is significantly more stable both in vitro and in vivo. Compound <b>67</b> is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1–UBE2M interaction on the NEDD8/CUL pathway

Discovery of an Orally Bioavailable Inhibitor of Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously reported the discovery, validation, and structure–activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1–UBE2M interaction. We demonstrated that compound <b>7</b> inhibits both the DCN1–UBE2M protein–protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound <b>7</b> exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CL_int = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (<b>67</b>). Compound <b>67</b> retains the favorable biochemical and cellular activity of compound <b>7</b> but is significantly more stable both in vitro and in vivo. Compound <b>67</b> is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1–UBE2M interaction on the NEDD8/CUL pathway

Discovery of an Orally Bioavailable Inhibitor of Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously reported the discovery, validation, and structure–activity relationships of a series of piperidinyl ureas that potently inhibit the DCN1–UBE2M interaction. We demonstrated that compound <b>7</b> inhibits both the DCN1–UBE2M protein–protein interaction and DCN1-mediated cullin neddylation in biochemical assays and reduces levels of steady-state cullin neddylation in a squamous carcinoma cell line harboring DCN1 amplification. Although compound <b>7</b> exhibits good solubility and permeability, it is rapidly metabolized in microsomal models (CL_int = 170 mL/min/kg). This work lays out the discovery of an orally bioavailable analogue, NAcM-OPT (<b>67</b>). Compound <b>67</b> retains the favorable biochemical and cellular activity of compound <b>7</b> but is significantly more stable both in vitro and in vivo. Compound <b>67</b> is orally bioavailable, well tolerated in mice, and currently used to study the effects of acute pharmacologic inhibition of the DCN1–UBE2M interaction on the NEDD8/CUL pathway

Piperidinyl Ureas Chemically Control Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure–activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1–UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1–UBE2M protein–protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers

Piperidinyl Ureas Chemically Control Defective in Cullin Neddylation 1 (DCN1)-Mediated Cullin Neddylation

We previously discovered and validated a class of piperidinyl ureas that regulate defective in cullin neddylation 1 (DCN1)-dependent neddylation of cullins. Here, we report preliminary structure–activity relationship studies aimed at advancing our high-throughput screen hit into a tractable tool compound for dissecting the effects of acute DCN1–UBE2M inhibition on the NEDD8/cullin pathway. Structure-enabled optimization led to a 100-fold increase in biochemical potency and modestly increased solubility and permeability as compared to our initial hit. The optimized compounds inhibit the DCN1–UBE2M protein–protein interaction in our TR-FRET binding assay and inhibit cullin neddylation in our pulse-chase NEDD8 transfer assay. The optimized compounds bind to DCN1 and selectively reduce steady-state levels of neddylated CUL1 and CUL3 in a squamous cell carcinoma cell line. Ultimately, we anticipate that these studies will identify early lead compounds for clinical development for the treatment of lung squamous cell carcinomas and other cancers