Development of Small Molecules that Specifically Inhibit the D‑loop Activity of RAD51

RAD51 is the central protein in homologous recombination (HR) DNA repair and represents a therapeutic target in oncology. Herein we report a novel class of RAD51 inhibitors that were identified by high throughput screening. In contrast to many previously reported RAD51 inhibitors, our lead compound <b>1</b> is capable of blocking RAD51-mediated D-loop formation (IC₅₀ 21.3 ± 7.8 μM) at concentrations that do not influence RAD51 binding to ssDNA. In human cells, <b>1</b> inhibits HR (IC₅₀ 13.1 ± 1.6 μM) without blocking RAD51’s ability to assemble into subnuclear foci at sites of DNA damage. We determined that the active constituent of <b>1</b> is actually an oxidized derivative (termed <b>RI­(dl)-1</b> or <b>8</b>) of the original screening compound. Our SAR campaign also yielded <b>RI­(dl)-2</b> (hereafter termed <b>9h</b>), which effectively blocks RAD51’s D-loop activity in biochemical systems (IC₅₀ 11.1 ± 1.3 μM) and inhibits HR activity in human cells (IC₅₀ 3.0 ± 1.8 μM)

Design and Synthesis of Mercaptoacetamides as Potent, Selective, and Brain Permeable Histone Deacetylase 6 Inhibitors

A series of nonhydroxamate HDAC6 inhibitors were prepared in our effort to develop potent and selective compounds for possible use in central nervous system (CNS) disorders, thus obviating the genotoxicity often associated with the hydroxamates. Halogens are incorporated in the cap groups of the designed mercaptoacetamides in order to increase brain accessibility. The indole analogue <b>7e</b> and quinoline analogue <b>13a</b> displayed potent HDAC6 inhibitory activity (IC₅₀, 11 and 2.8 nM) and excellent selectivity against HDAC1. Both <b>7e</b> and <b>13a</b> together with their ester prodrug <b>14</b> and disulfide prodrugs <b>15</b> and <b>16</b> were found to be effective in promoting tubulin acetylation in HEK cells. The disulfide prodrugs <b>15</b> and <b>16</b> also released a stable concentration of <b>7e</b> and <b>13a</b> upon microsomal incubation. Administration of <b>15</b> and <b>16</b> <i>in vivo</i> was found to trigger an increase of tubulin acetylation in mouse cortex. These results suggest that further exploration of these compounds for the treatment of CNS disorders is warranted

Discovery of <i>N</i>‑Substituted (2-Phenylcyclopropyl)methylamines as Functionally Selective Serotonin 2C Receptor Agonists for Potential Use as Antipsychotic Medications

A series of <i>N</i>-substituted (2-phenylcyclopropyl)­methylamines were designed and synthesized, with the aim of finding serotonin 2C (5-HT_2C)-selective agonists with a preference for G_q signaling. A number of these compounds exhibit 5-HT_2C selectivity with a preference for G_q-mediated signaling compared with β-arrestin recruitment. Furthermore, the <i>N-</i>methyl compound (+)-<b>15a</b>, which displayed an EC₅₀ of 23 nM in the calcium flux assay while showing no β-arrestin recruitment activity, is the most functionally selective 5-HT_2C agonist reported to date. The <i>N</i>-benzyl compound (+)-<b>19</b>, which showed an EC₅₀ of 24 nM at the 5-HT_2C receptor, is fully selective over the 5-HT_2B receptor. In an amphetamine-induced hyperactivity model, compound (+)-<b>19</b> showed significant antipsychotic-drug-like activity. These novel compounds shed light on the role of functional selectivity at the 5-HT_2C receptor with respect to antipsychotic activity

Thiol-Based Potent and Selective HDAC6 Inhibitors Promote Tubulin Acetylation and T‑Regulatory Cell Suppressive Function

Several new mercaptoacetamides were synthesized and studied as HDAC6 inhibitors. One compound, <b>2b</b>, bearing an aminoquinoline cap group, was found to show 1.3 nM potency at HDAC6, with >3000-fold selectivity over HDAC1. <b>2b</b> also showed excellent efficacy at increasing tubulin acetylation in rat primary cortical cultures, inducing a 10-fold increase in acetylated tubulin at 1 μM. To assess possible therapeutic effects, compounds were assayed for their ability to increase T-regulatory (Treg) suppressive function. Some but not all of the compounds increased Treg function, and thereby decreased conventional T cell activation and proliferation <i>in vitro</i>

An Optimized RAD51 Inhibitor That Disrupts Homologous Recombination without Requiring Michael Acceptor Reactivity

Homologous recombination (HR) is an essential process in cells that provides repair of DNA double-strand breaks and lesions that block DNA replication. RAD51 is an evolutionarily conserved protein that is central to HR. Overexpression of RAD51 protein is common in cancer cells and represents a potential therapeutic target in oncology. We previously described a chemical inhibitor of RAD51, called RI-1 (referred to as compound <b>1</b> in this report). The chloromaleimide group of this compound is thought to act as a Michael acceptor and react with the thiol group on C319 of RAD51, using a conjugate addition–elimination mechanism. In order to reduce the likelihood of off-target effects and to improve compound stability in biological systems, we developed an analogue of compound <b>1</b> that lacks maleimide-based reactivity but retains RAD51 inhibitory activity. This compound, 1-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)-4-morpholino-1<i>H</i>-pyrrole-2,5-dione, named RI-2 (referred to as compound <b>7a</b> in this report), appears to bind reversibly to the same site on the RAD51 protein as does compound <b>1</b>. Like compound <b>1</b>, compound <b>7a</b> specifically inhibits HR repair in human cells

Synthesis and Pharmacological Evaluation of Selective Histone Deacetylase 6 Inhibitors in Melanoma Models

Only a handful of therapies offer significant improvement in the overall survival in cases of melanoma, a cancer whose incidence has continued to rise in the past 30 years. In our effort to identify potent and isoform-selective histone deacetylase (HDAC) inhibitors as a therapeutic approach to melanoma, a series of new HDAC6 inhibitors based on the nexturastat A scaffold were prepared. The new analogues <b>4d</b>, <b>4e</b>, and <b>7b</b> bearing added hydrophilic substituents, so as to establish additional hydrogen bonding on the rim of the HDAC6 catalytic pocket, exhibit improved potency against HDAC6 and retain selectivity over HDAC1. Compound <b>4d</b> exhibits antiproliferative effects on several types of melanoma and lymphoma cells. Further studies indicates that <b>4d</b> selectively increases acetylated tubulin levels <i>in vitro</i> and elicits an immune response through down-regulating cytokine IL-10. A preliminary <i>in vivo</i> efficacy study indicates that <b>4d</b> possesses improved capability to inhibit melanoma tumor growth and that this effect is based on the regulation of inflammatory and immune responses

Design and Discovery of Functionally Selective Serotonin 2C (5-HT_2C) Receptor Agonists

On the basis of the structural similarity of our previous 5-HT_2C agonists with the melatonin receptor agonist tasimelteon and the putative biological cross-talk between serotonergic and melatonergic systems, a series of new (2,3-dihydro)­benzofuran-based compounds were designed and synthesized. The compounds were evaluated for their selectivity toward 5-HT_2A, 5-HT_2B, and 5-HT_2C receptors in the calcium flux assay with the ultimate goal to generate selective 5-HT_2C agonists. Selected compounds were studied for their functional selectivity by comparing their transduction efficiency at the G protein signaling pathway versus β-arrestin recruitment. The most functionally selective compound (+)-<b>7e</b> produced weak β-arrestin recruitment and also demonstrated less receptor desensitization compared to serotonin in both calcium flux and phosphoinositide (PI) hydrolysis assays. We report for the first time that selective 5-HT_2C agonists possessing weak β-arrestin recruitment can produce distinct receptor desensitization properties

Indole-2-carboxamide-based MmpL3 Inhibitors Show Exceptional Antitubercular Activity in an Animal Model of Tuberculosis Infection

Our team had previously identified certain indolecarboxamides that represented a new chemical scaffold that showed promising anti-TB activity at both an <i>in vitro</i> and <i>in vivo</i> level. Based on mutational analysis using bacteria found resistant to one of these indolecarboxamides, we identified the trehalose monomycolate transporter MmpL3 as the likely target of these compounds. In the present work, we now further elaborate on the SAR of these compounds, which has led in turn to the identification of a new analog, 4,6-difluoro-<i>N</i>-((1<i>R</i>,2<i>R</i>,3<i>R</i>,5<i>S</i>)-2,6,6-trimethylbicyclo­[3.1.1]­heptan-3-yl)-1<i>H</i>-indole-2-carboxamide (<b>26</b>), that shows excellent activity against drug-sensitive (MIC = 0.012 μM; SI ≥ 16000), multidrug-resistant (MDR), and extensively drug-resistant (XDR) <i>Mycobacterium tuberculosis</i> strains, has superior ADMET properties, and shows excellent activity in the TB aerosol lung infection model. Compound <b>26</b> is also shown to work in synergy with rifampin. Because of these properties, we believe that indolecarboxamide <b>26</b> is a possible candidate for advancement to human clinical trials

Identification of a Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitor, BIP-135, That Prolongs the Median Survival Time of Δ7 SMA KO Mouse Model of Spinal Muscular Atrophy

The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by Western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration

Indole-2-carboxamide-based MmpL3 Inhibitors Show Exceptional Antitubercular Activity in an Animal Model of Tuberculosis Infection

Our team had previously identified certain indolecarboxamides that represented a new chemical scaffold that showed promising anti-TB activity at both an <i>in vitro</i> and <i>in vivo</i> level. Based on mutational analysis using bacteria found resistant to one of these indolecarboxamides, we identified the trehalose monomycolate transporter MmpL3 as the likely target of these compounds. In the present work, we now further elaborate on the SAR of these compounds, which has led in turn to the identification of a new analog, 4,6-difluoro-<i>N</i>-((1<i>R</i>,2<i>R</i>,3<i>R</i>,5<i>S</i>)-2,6,6-trimethylbicyclo­[3.1.1]­heptan-3-yl)-1<i>H</i>-indole-2-carboxamide (<b>26</b>), that shows excellent activity against drug-sensitive (MIC = 0.012 μM; SI ≥ 16000), multidrug-resistant (MDR), and extensively drug-resistant (XDR) <i>Mycobacterium tuberculosis</i> strains, has superior ADMET properties, and shows excellent activity in the TB aerosol lung infection model. Compound <b>26</b> is also shown to work in synergy with rifampin. Because of these properties, we believe that indolecarboxamide <b>26</b> is a possible candidate for advancement to human clinical trials