The Parallel Structure-Activity Relationship Screening of Three Compounds Identifies the Common Agonist Pharmacophore of Pyrrolidine Bis-Cyclic Guanidine Melanocortin-3 Receptor (MC3R) Small-Molecule Ligands.

The melanocortin receptors are involved in numerous physiological pathways, including appetite, skin and hair pigmentation, and steroidogenesis. In particular, the melanocortin-3 receptor (MC3R) is involved in fat storage, food intake, and energy homeostasis. Small-molecule ligands developed for the MC3R may serve as therapeutic lead compounds for treating disease states of energy disequilibrium. Herein, three previously reported pyrrolidine bis-cyclic guanidine compounds with five sites for molecular diversity (R1-R5) were subjected to parallel structure-activity relationship studies to identify the common pharmacophore of this scaffold series required for full agonism at the MC3R. The R2, R3, and R5 positions were required for full MC3R efficacy, while truncation of either the R1 or R4 positions in all three compounds resulted in full MC3R agonists. Two additional fragments, featuring molecular weights below 300 Da, were also identified that possessed full agonist efficacy and micromolar potencies at the mMC5R. These SAR experiments may be useful in generating new small-molecule ligands and chemical probes for the melanocortin receptors to help elucidate their roles in vivo and as therapeutic lead compounds.

Amide- and bis-amide-linked highly potent and broadly active antifungal agents for the treatment of invasive fungal infections- towards the discovery of pre-clinical development candidate FC12406.

Most fungal infections are common, localized to skin or mucosal surfaces and can be treated effectively with topical antifungal agents. However, while invasive fungal infections (IFIs) are uncommon, they are very difficult to control medically, and are associated with high mortality rates. We have previously described highly potent bis-guanidine-containing heteroaryl-linked antifungal agents, and were interested in expanding the range of agents to novel series so as to reduce the degree of aromaticity (with a view to making the compounds more drug-like), and provide broadly active high potency derivatives. We have investigated the replacement of the central aryl ring from our original series by both amide and a bis-amide moieties, and have found particular structure-activity relationships (SAR) for both series', resulting in highly active antifungal agents against both mold and yeast pathogens. In particular, we describe the in vitro antifungal activity, absorption, distribution, metabolism and elimination (ADME) properties, and off-target properties of FC12406 (34), which was selected as a pre-clinical development candidate.

Atypical chorioretinal lesions in Siberian Husky dogs with primary angle-closure glaucoma: a case series.

BACKGROUND: A number of etiologies for different canine chorioretinal lesions have been proved or suggested but some fundic lesions remain unclear in terms of an etiologic diagnosis, treatment options and prognosis. The purpose of this case series is to describe atypical chorioretinal lesions observed in dogs with primary angle-closure glaucoma (PACG). CASE PRESENTATION: Two spayed-female Siberian Huskies (3- and 4-year-old) and one Siberian Husky/Australian Shepherd mixed breed dog (11-month-old) that had multifocal depigmented retinal lesions and PACG were included. PROCEDURES: Ophthalmic examination, gross, and histopathologic examination findings are described. One of the dogs underwent further clinical diagnostics. Advanced clinical diagnostics on the fellow, presumed to be non-glaucomatous eye of a dog revealed: pectinate ligament dysplasia by gonioscopy, retinal thinning in the depigmented area and wedge shaped retinal thinning with delayed choroidal vascular perfusion by optical coherence tomography, confocal scanning laser ophthalmoscopy, fluorescein and indocyanine green angiography. Quantifiable maze testing for the same eye revealed mild nyctalopia but the full-field electroretinogram showed no generalized decrease of retinal function. Genetic testing for mutations within the retinitis pigmentosa GTPase regulator gene causing X-linked progressive retinal atrophy in Siberian Huskies was negative. Histopathologic evaluations on enucleated eyes in two dogs confirmed goniodysgenesis, PACG with optic nerve head cupping, and diffuse inner retinal atrophy. In addition, segmental profound retinal atrophy, loss of retinal pigment epithelium, and adhesion of the retina to Bruch's membrane was observed and coincided with multifocal depigmented lesions noted on fundic examination. CONCLUSIONS: To our knowledge, this is the first case series with clinical and histopathologic data of chorioretinal lesions, most likely caused by severely impaired choroidal perfusion. Further studies are warranted to elucidate the etiology and pathophysiology, including its possible association with PACG.

Highly potent, broadly active antifungal agents for the treatment of invasive fungal infections.

Invasive fungal infections have become an important healthcare issue due in large part to high mortality rates under standard of care (SOC) therapies creating an urgent need for new and effective anti-fungal agents. We have developed a series of non-peptide, structurally-constrained analogs of host defence proteins that have distinct advantages over peptides for pharmaceutical uses. Here we report the chemical optimization of bis-guanidine analogs focused on alterations of the central aryl core and the connection of it to the terminal guanidines. This effort resulted in the production of highly potent, broadly active compounds with low mammalian cell cytotoxicity that have comparable or improved antifungal activities over SOC agents. One optimal compound was also found to possess favourable in vitro pharmaceutical and off-target properties suitable for further development.

RUNX3 levels in human hematopoietic progenitors are regulated by aging and dictate erythroid-myeloid balance.

Healthy bone marrow progenitors yield a co-ordinated balance of hematopoietic lineages. This balance shifts with aging toward enhanced granulopoiesis with diminished erythropoiesis and lymphopoiesis, changes which likely contribute to the development of bone marrow disorders in the elderly. In this study, RUNX3 was identified as a hematopoietic stem and progenitor cell factor whose levels decline with aging in humans and mice. This decline is exaggerated in hematopoietic stem and progenitor cells from subjects diagnosed with unexplained anemia of the elderly. Hematopoietic stem cells from elderly unexplained anemia patients had diminished erythroid but unaffected granulocytic colony forming potential. Knockdown studies revealed human hematopoietic stem and progenitor cells to be strongly influenced by RUNX3 levels, with modest deficiencies abrogating erythroid differentiation at multiple steps while retaining capacity for granulopoiesis. Transcriptome profiling indicated control by RUNX3 of key erythroid transcription factors, including KLF1 and GATA1 These findings thus implicate RUNX3 as a participant in hematopoietic stem and progenitor cell aging, and a key determinant of erythroid-myeloid lineage balance.

Structure⁻Activity Relationships of the Tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 at the Mouse Melanocortin Receptors: Modification at the (pI)DPhe Position Leads to mMC3R Versus mMC4R Selective Ligands.

The five melanocortin receptors (MC1R-MC5R) are involved in numerous biological pathways, including steroidogenesis, pigmentation, and food intake. In particular, MC3R and MC4R knockout mice suggest that the MC3R and MC4R regulate energy homeostasis in a non-redundant manner. While MC4R-selective agonists have been utilized as appetite modulating agents, the lack of MC3R-selective agonists has impeded progress in modulating this receptor in vivo. In this study, the (pI)DPhe position of the tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 (an MC3R agonist/MC4R antagonist ligand) was investigated with a library of 12 compounds. The compounds in this library were found to have higher agonist efficacy and potency at the mouse (m) MC3R compared to the MC4R, indicating that the Arg-DPhe motif preferentially activates the mMC3R over the mMC4R. This observation may be used in the design of new MC3R-selective ligands, leading to novel probe and therapeutic lead compounds that will be useful for treating metabolic disorders.

A fragment of the Escherichia coli ClpB heat-shock protein is a micromolar melanocortin 1 receptor agonist.

The melanocortin system consists of five receptor subtypes (MC1-5R), endogenous agonists derived from the proopiomelanocortin gene transcript, and the antagonists agouti and agouti-related protein. The Escherichia coli heat shock protein ClpB has previously been described as an antigen mimetic to the endogenous melanocortin agonist α-MSH. Herein, we investigated if a fragment of the ClpB protein could directly signal through the melanocortin receptors. We synthesized a complementary fragment of the ClpB protein that partially aligned with α-MSH. Pharmacological assessment of this fragment resulted in no antagonist activity at the MC3R or the MC4R and no agonist activity at the MC4R. Partial receptor activation was observed for the MC3R and MC5R at 100 µM concentrations. This fragment was shown to be a full micromolar MC1R agonist and may serve as a template for future research into selective MC1R ligands.

Acinetobacter baumannii OxPhos inhibitors as selective anti-infective agents.

The Gram-negative bacterium Acinetobacter baumannii is an opportunistic pathogen in humans and infections are poorly treated by current therapy. Recent emergence of multi-drug resistant strains and the lack of new antibiotics demand an immediate action for development of new anti-Acinetobacter agents. To this end, oxidative phosphorylation (OxPhos) was identified as a novel target for drug discovery research. Consequently, a library of ∼10,000 compounds was screened using a membrane-based ATP synthesis assay. One hit identified was the 2-iminobenzimidazole 1 that inhibited the OxPhos of A. baumannii with a modestly high selectivity against mitochondrial OxPhos, and displayed an MIC of 25µM (17µg/mL) against the pathogen. The 2-iminobenzimidazole 1 was found to inhibit the type 1 NADH-quinone oxidoreductase (NDH-1) of A. baumannii OxPhos by a biochemical approach. Among various derivatives that were synthesized to date, des-hydroxy analog 5 is among the most active with a relatively tight SAR requirement for the N'-aminoalkyl side chain. Analog 5 also showed less cytotoxicity against NIH3T3 and HepG2 mammalian cell lines, demonstrating the potential for this series of compounds as anti-Acinetobacter agents. Additional SAR development and target validation is underway.

Evaluation of viruses and their association with ocular lesions in pinnipeds in rehabilitation.

OBJECTIVE: To assess whether corneal lesions in stranded pinnipeds were associated with viral infections, and to identify the potential pathogen(s) associated with the lesions. ANIMALS STUDIED: Twenty-nine California sea lions (Zalophus californianus), 18 northern elephant seals (Mirounga angustirostris), and 34 Pacific harbor seals (Phoca vitulina richardsii). PROCEDURE: DNA and RNA were extracted from ocular swabs, corneal tissue, and aqueous humor and screened for herpesvirus, adenovirus, poxvirus, and calicivirus families by PCR. RESULTS: The results indicated a high overall prevalence of viruses, with adenoviruses and herpesviruses detected in all three host species. Three novel adenoviruses (PhAdV-1, PhAdV-2, OtAdV-2) and two novel herpesviruses (PhHV-6, OtHV-4) were detected. There were no statistical differences in the prevalence of viral infection or coinfection among groups of individuals with or without corneal lesions, nor were lesion type, onset, or presence of concurrent disease significantly associated with a viral infection. CONCLUSIONS: The results suggested that viral presence in ocular tissues was common, not significantly associated with ocular disease and thus should not preclude release of an otherwise healthy animal. We could not confirm a correlation of virus presence with lesion due to the high percentage of virus-positive, clinically normal animals. This implied that seals and sea lions can have ocular tissues infected with several viruses without having readily evident associated lesions. This difficulty in correlating viral presence, particularly herpesviruses, with ocular lesions was also a common finding in studies with terrestrial species and highlighted the difficulty of confirming a virus as a primary pathogen in ocular lesions.

Activity of potent and selective host defense peptide mimetics in mouse models of oral candidiasis.

There is a strong need for new broadly active antifungal agents for the treatment of oral candidiasis that not only are active against many species of Candida, including drug-resistant strains, but also evade microbial countermeasures which may lead to resistance. Host defense peptides (HDPs) can provide a foundation for the development of such agents. Toward this end, we have developed fully synthetic, small-molecule, nonpeptide mimetics of the HDPs that improve safety and other pharmaceutical properties. Here we describe the identification of several HDP mimetics that are broadly active against C. albicans and other species of Candida, rapidly fungicidal, and active against yeast and hyphal cultures and that exhibit low cytotoxicity for mammalian cells. Importantly, specificity for Candida over commensal bacteria was also evident, thereby minimizing potential damage to the endogenous microbiome which otherwise could favor fungal overgrowth. Three compounds were tested as topical agents in two different mouse models of oral candidiasis and were found to be highly active. Following single-dose administrations, total Candida burdens in tongues of infected animals were reduced up to three logs. These studies highlight the potential of HDP mimetics as a new tool in the antifungal arsenal for the treatment of oral candidiasis.

Incorporation of Three Extracyclic Arginine Residues into a Melanocortin Macrocyclic Agonist (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Arg-Arg-Arg-Ac)-DPro]) Decreases Food Intake When Administered Intrathecally or Subcutaneously Compared to a Macrocyclic Ligand Lacking Extracyclic Arginine Residues (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro)].

Of the three Food and Drug Administration-approved melanocortin peptide drugs, two possess a cyclic scaffold, demonstrating that cyclized melanocortin peptides have therapeutic relevance. An extracyclic Arg residue, critical for pharmacological activity in the approved melanocortin cyclic drug setmelanotide, has also been demonstrated to increase the signal when fluorescently labeled cell-penetrating cyclic peptides are incubated with HeLa cells, with the maximal signal observed with three extracyclic Arg amino acids. Herein, a branching Lys residue was substituted into two macrocyclic melanocortin peptide agonists to incorporate 0-3 extracyclic Arg amino acids. Incorporation of the Arg residues resulted in equipotent or increased agonist potency at the mouse melanocortin receptors in vitro, suggesting that these substitutions were tolerated in the macrocyclic scaffolds. Further in vivo evaluation of one parent ligand (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-Pro]) and the three Arg derivative (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Ac-Arg-Arg-Arg)-Pro)] demonstrated that the three Arg derivative further decreased food intake compared to the parent macrocycle when the compounds were administered either via intrathecal injection or subcutaneous dosing. This suggests that three extracyclic Arg amino acids may be beneficial in the design of cyclic melanocortin ligands and that in vitro pharmacological profiling may not predict the in vivo efficacy of melanocortin ligands.

Assessing Longitudinal Treatment Efficacies and Alterations in Molecular Markers Associated with Glutamatergic Signaling and Immune Checkpoint Inhibitors in a Spontaneous Melanoma Mouse Model.

Previous work done by our laboratory described the use of an immunocompetent spontaneous melanoma-prone mouse model, TGS (TG-3/SKH-1), to evaluate treatment outcomes using inhibitors of glutamatergic signaling and immune checkpoint for 18 weeks. We showed a significant therapeutic efficacy with a notable sex-biased response in male mice. In this follow-up 18-week study, the dose of the glutamatergic signaling inhibitor was increased (from 1.7 mg/kg to 25 mg/kg), which resulted in improved responses in female mice but not male mice. The greatest reduction in tumor progression was observed in male mice treated with single-agent troriluzole and anti-PD-1. Furthermore, a randomly selected group of mice was removed from treatment after 18 weeks and maintained for up to an additional 48 weeks demonstrating the utility of the TGS mouse model to perform a ≥1-year preclinical therapeutic study in a physiologically relevant tumor-host environment. Digital spatial imaging analyses were performed in tumors and tumor microenvironments across treatment modalities using antibody panels for immune cell types and immune cell activation. The results suggest that immune cell populations and cytotoxic activities of T cells play critical roles in treatment responses in these mice. Examination of a group of molecular protein markers based on the proposed mechanisms of action of inhibitors of glutamatergic signaling and immune checkpoint showed that alterations in expression levels of xCT, γ-H2AX, EAAT2, PD-L1, and PD-1 are likely associated with the loss of treatment responses. These results suggest the importance of tracking changes in molecular markers associated with the mechanism of action of therapeutics over the course of a longitudinal preclinical therapeutic study in spatial and temporal manners.

Neuroplasticity of sensory and sympathetic nerve fibers in a mouse model of a painful arthritic joint.

OBJECTIVE: Many forms of arthritis are accompanied by significant chronic joint pain. This study was undertaken to investigate whether there is significant sprouting of sensory and sympathetic nerve fibers in the painful arthritic knee joint and whether nerve growth factor (NGF) drives this pathologic reorganization. METHODS: A painful arthritic knee joint was produced by injection of Freund's complete adjuvant (CFA) into the knee joint of young adult mice. CFA-injected mice were then treated systemically with vehicle or anti-NGF antibody. Pain behaviors were assessed, and at 28 days following the initial CFA injection, the knee joints were processed for immunohistochemistry analysis using antibodies against calcitonin gene-related peptide (CGRP; sensory nerve fibers), neurofilament 200 kd (NF200; sensory nerve fibers), growth-associated protein 43 (GAP-43; sprouted nerve fibers), tyrosine hydroxylase (TH; sympathetic nerve fibers), CD31 (endothelial cells), or CD68 (monocyte/macrophages). RESULTS: In CFA-injected mice, there was a significant increase in the density of CD68+ macrophages, CD31+ blood vessels, and CGRP+, NF200+, GAP-43+, and TH+ nerve fibers in the synovium, as well as a significant increase in joint pain-related behaviors. None of these findings were observed in sham-injected mice. Administration of anti-NGF reduced these pain-related behaviors and the ectopic sprouting of nerve fibers, but had no significant effect on the increase in density of CD31+ blood vessels or CD68+ macrophages. CONCLUSION: These findings demonstrate that ectopic sprouting of sensory and sympathetic nerve fibers occurs in the painful arthritic joint and may be involved in the generation and maintenance of arthritic pain.

Discovery of a Pan-Melanocortin Receptor Antagonist [Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] at the MC1R, MC3R, MC4R, and MC5R that Mediates an Increased Feeding Response in Mice and a 40-Fold Selective MC1R Antagonist [Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2].

Discovery of pan-antagonist ligands for the melanocortin receptors will help identify the physiological activities controlled by these receptors. The previously reported MC3R/MC4R antagonist Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2 was identified herein, for the first time, to possess MC1R and MC5R antagonist activity. Further structure-activity relationship studies probing the second and fourth positions were performed toward the goal of identifying potent melanocortin antagonists. Of the 21 tetrapeptides synthesized, 13 possessed MC1R, MC3R, MC4R, and MC5R antagonist activity. Three tetrapeptides were more than 10-fold selective for the mMC1R, including 8 (LTT1-44, Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2) that possessed 80 nM mMC1R antagonist potency and was at least 40-fold selective over the mMC3R, mMC4R, and mMC5R. Nine tetrapeptides were selective for the mMC4R, including 14 [SSM1-8, Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] with an mMC4R antagonist potency of 1.6 nM. This compound was administered IT into mice, resulting in a dose-dependent increase in the food intake and demonstrating the in vivo utility of this compound series.

A Spontaneous Melanoma Mouse Model Applicable for a Longitudinal Chemotherapy and Immunotherapy Study.

Mouse models that reflect human disorders provide invaluable tools for the translation of basic science discoveries to clinical therapies. However, many of these in vivo therapeutic studies are short term and do not accurately mimic patient conditions. In this study, we used a fully immunocompetent, transgenic mouse model, TGS, in which the spontaneous development of metastatic melanoma is driven by the ectopic expression of a normal neuronal receptor, mGluR1, as a model to assess longitudinal treatment response (up to 8 months) with an inhibitor of glutamatergic signaling, troriluzole, which is a prodrug of riluzole, plus an antibody against PD-1, an immune checkpoint inhibitor. Our results reveal a sex-biased treatment response that led to improved survival in troriluzole and/or anti-PD-1-treated male mice that correlated with differential CD8+ T cells and CD11b+ myeloid cell populations in the tumor-stromal interface, supporting the notion that this model is a responsive and tractable system for evaluating therapeutic regimens for melanoma in an immunocompetent setting.

Incorporation of Indoylated Phenylalanine Yields a Sub-Micromolar Selective Melanocortin-4 Receptor Antagonist Tetrapeptide.

The melanocortin family is involved in many physiological functions, including pigmentation, steroidogenesis, and appetite. The centrally expressed melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R) possess overlapping but distinct roles in energy homeostasis. Herein, the third and fourth positions of a tetrapeptide lead compound [Ac-Arg-Arg-(pI)DPhe-Tic-NH2], previously reported to possess MC3R agonist and MC4R antagonist activities, were substituted with indoylated phenylalanine (Wsf/Wrf) residues in an attempt to generate receptor subtype selective compounds. At the third position, d-amino acids were required for melanocortin agonist activity, while both l- and d-residues resulted in MC4R antagonist activity. These results indicate that l-indoylated phenylalanine residues at the third position of the scaffold can generate MC4R over MC3R selective antagonist ligands, resulting in a substitution pattern that may be exploited for novel MC4R ligands that can be used to probe the in vivo activity of the MC4R without involvement of the MC3R.

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

Pathological sprouting of adult nociceptors in chronic prostate cancer-induced bone pain.

Pain frequently accompanies cancer. What remains unclear is why this pain frequently becomes more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the tumor-bearing tissue undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of canine prostate cancer cells into mouse bone induces a remarkable sprouting of calcitonin gene-related peptide (CGRP(+)) and neurofilament 200 kDa (NF200(+)) sensory nerve fibers. Nearly all sensory nerve fibers that undergo sprouting also coexpress tropomyosin receptor kinase A (TrkA(+)). This ectopic sprouting occurs in sensory nerve fibers that are in close proximity to colonies of prostate cancer cells, tumor-associated stromal cells and newly formed woven bone, which together form sclerotic lesions that closely mirror the osteoblastic bone lesions induced by metastatic prostate tumors in humans. Preventive treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. Interestingly, reverse transcription PCR analysis indicated that the prostate cancer cells themselves do not express detectable levels of mRNA coding for NGF. This suggests that the tumor-associated stromal cells express and release NGF, which drives the pathological reorganization of nearby TrkA(+) sensory nerve fibers. Therapies that prevent this reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive cancer pain and lead to more effective control of this chronic pain state.

Capsular Management Techniques and Hip Arthroscopy.

There has been an increased emphasis on capsular management during hip arthroscopy in the literature in recent years. The capsule plays a significant role in the hip joint stability and studies have demonstrated that capsular closure can restore the biomechanics of the hip back to the native state. Capsular management also affects functional outcomes with capsular repair resulting in better clinical outcomes in some studies. Management of the capsule has evolved in recent years with more surgeons performing routine capsular closure. Management techniques and degree of capsular closure, however, can be quite variable between surgeons. This review will discuss hip capsular anatomy, the importance of the capsule in hip biomechanics, management of the capsule during arthroscopy, and functional outcomes as it relates to the various capsular closure techniques versus leaving the capsulotomy unrepaired.

Multiresidue Tetrapeptide Substitutions Yield a 140-fold Selective Melanocortin-3 over Melanocortin-4 Receptor Agonist.

The five melanocortin receptors regulate numerous physiological functions. Although many ligands have been developed for the melanocortin-4 receptor (MC4R), the melanocortin-3 receptor (MC3R) has been less-well characterized, in part due to the lack of potent, selective tool compounds. Previously an Ac-His-Arg-(pI)DPhe-Tic-NH2 scaffold, inverting the Phe-Arg motif of the native melanocortin signal sequence, was identified to possess mMC3R over mMC4R selective agonist activity. In this study, a library of 12 compounds derived from this scaffold was synthesized and assayed at the mouse melanocortin receptors (MCRs), utilizing substitutions previously shown to increase mMC3R agonist potency and/or selectivity. One compound (8, Ac-Val-Gln-DBip-DTic-NH2) was identified as greater than 140-fold selective for the mMC3R over the mMC4R, possessed 70 nM potency at the mMC3R, and partially stimulated the mMC4R at 100 µM concentrations without antagonist activity. This pharmacological profile may be useful in developing new tool and therapeutic ligands that selective signal through the MC3R.