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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

ML302, a Novel Beta-lactamase (BLA) Inhibitor

Authors

,1 ,1 ,1 ,1 ,2 ,2 ,1 ,2 and 1,3.

Affiliations

1 Lead Identification Division, Translational Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Fl, 33458
2 Department of Chemistry, Scripps Florida, 130 Scripps Way, Jupiter, Fl, 33458
3 To whom correspondence should be addressed, ude.sppircs@preddoh

Received: ; Last Update: May 13, 2014.

VIM-2 and IMP-1 are Ambler class B metallo-β-lactamases (MBL) capable of hydrolyzing a broad-spectrum of β-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, non-selective (broad-acting) small molecule inhibitors is yet to be fully characterized. Here we describe a novel dual VIM-2/IMP-1 inhibitor that exhibits efficacy in multiple clinical isolates. We therefore claim CID 53362017/SID 134220672 as a potent, broad acting VIM-2/IMP-1 probe (ML302). This compound was discovered from a medicinal chemistry effort that sought to improve the potency and efficacy of high-throughput screening (HTS) hits. During these chemistry efforts, we identified a rhodanine scaffold that exhibited activity against recombinant VIM-2 and IMP-1 in nitrocefin-based enzyme activity assays. Various secondary assays were run to determine its dual potency (VIM-2 IC50 = 548 nM; IMP-1 IC50 = 3.02 μM) and class B selectivity (it was inactive in TEM-1 and AmpC beta-lactamase enzymatic assays). Kinetic analyses demonstrated that ML302 behaves as a mixed mode uncompetitive/non-competitive inhibitor, with submicromolar Ki values against VIM-2 and IMP-1 (Ki = 183 ± 24 nM and 930 ± 97 nM, respectively). This represents an improvement in both our prior probe ML121 and the previously existing art compound Mitoxantrone, each of which inhibited only VIM-2. Subsequent studies revealed that this probe potentiates the activity of imipenem antibiotic in inhibiting growth of laboratory E. coli BL21 strains harboring VIM-2 and IMP-1, as well as clinical isolates YMC07 (VIM-2-containing Acinetobacter sp.), BAA-2146 (NDM-1-containing Klebsiella pneumonia), PA641 (VIM-2-containing Pseudomonas aeruginosa), and KN20 (IMP-1-containing Pseudomonas aeruginosa). This probe will serve as a valuable tool to elucidate the role of VIM-2 and IMP-1 in nosocomial beta-lactam antibiotic resistance.

Assigned Assay Grant #: 1 R21 NS059451-01 Fast Track

Screening Center Name & PI: Scripps Research Institute Molecular Screening Center (SRIMSC); Hugh Rosen

Chemistry Center Name & PI: SRIMSC; Hugh Rosen

Assay Submitter & Institution: Peter Hodder, TSRI

PubChem Summary Bioassay Identifier (AID): 1854

Probe Structure and Characteristics

DUAL VIM-2/IMP-1 Inhibitor Probe ML302.

DUAL VIM-2/IMP-1 Inhibitor Probe ML302

CID 53362017/SID 134220672

MLS003940491 (SR-030000025555)

CID/ML#Target NameIC50 (nM) [SID, AID]Anti-target NameIC50 (μM) [SID, AID]Fold SelectiveSecondary Assays: IC50 (nM) [SID, AID]
CID 53362017/ML302VIM-2Enzyme Assays:

IC50 = 548 nM [SID 125264855, AID 624079

Ki = 183 nM [SID134220672, AID 624083]		IMP-1Enzyme Assays:

IC50 = 3.018 μM [SID 125264855, AID 624085]

Ki = 930 nM [SID134220672, AID 624084]		>5.5Enzyme counterscreens:

TEM-1 IC50 >60 μM [SID125264855, AID 624092]
AmpC IC50 >60 μM [SID125264855, AID 624090]

Imipenem Synergy assays:
measured as a reduction (potentiation) of the MIC of imipenem in the following bacterial strains:

YMC07/8/B3323 (VIM-2)
Achieves 32X Potentiation at 781 nM [SID 125264855, AID 624097]
BL21 (VIM-2)
Achieves 8X Potentiation at 3.125 μM [SID 125264855, AID 624081]
PA641 (VIM-2)
Achieves 4X Potentiation at 50 μM [SID 125264855, AID 624080]
BAA-2146 (NDM-1)
Achieves 2X Potentiation at >50 μM [SID 125264855, AID 624082]
KN20 (IMP-1)
Achieves 2X Potentiation at >50 μM [SID 125264855, AID 624095]
BL21 (IMP-1)
Achieves 4X Potentiation at 781 nM [SID 125264855, AID 624097]

Recommendations for scientific use of the probe

Limitations in state of the art. We previously submitted a probe report describing a selective VIM-2 inhibitor (ML121). This probe was potent and active against the VIM-2 target. However, it lacked activity against the IMP-1 enzyme, which might limit its usefulness in clinically resistant bacterial strains that may express more than one β-lactamase. Another non-competitive VIM-2 inhibitor, mitoxantrone (1,4-dihydroxy-5,8-bis([2-([2-hydroxyethyl]amino)ethyl]amino)-9,10-anthracenedione; CID 4212), is a type II topoisomerase inhibitor that disrupts DNA synthesis and DNA repair in both healthy cells and cancer cells [1]. Like ML121, its inhibition is also selective to VIM-2 and its intense color limits Mitoxantrone’s use in certain assay detection formats. Another prior art compound, 4-chloromercuribenzoic acid (pCMB; CID 1730), is a slowly reversible/irreversible VIM-2 inhibitor shown to have a synergistic effect with β-lactam antibacterials in VIM-2-expressing bacteria.[2] Unfortunately, this cysteine-reactive reagent is known to have several off-target activities, lessening its value for mechanistic studies.

Probe Applications. Here we demonstrate that our probe ML302 (CID 53362017/SID 134220672/SR-03000002555) blocks the enzymatic activities of VIM-2 and IMP-1 in biochemical assays, with no apparent activity against class A (TEM-1) and class C (AmpC) b-lactamases. When dosed in clinical isolates, ML302 shows no toxicity; when co-dosed with imipenem, ML302 significantly increases imipenem’s efficacy (MIC). These findings have significant implications for studies that probe the enzymology of VIM-2 and IMP-1, especially mechanistic studies to better understand these enzymes’ broad-spectrum activity against various beta-lactam based antibiotics. Further, this probe can be useful for experiments that aim to inhibit VIM-2 and IMP-1 activity, without inhibiting TEM-1 or AmpC activity. In a broader role, the non-selectivity, potency, and efficacy of this compound will enable further investigations into the biological and biochemical roles of metallo-β-lactamase enzymes, and may be useful in the design of inhibitors to inhibit these clinically relevant enzymes and reduce the public health burden of antibiotic resistance.

Expected end-users of the probe in the research community. The probe can be used by researchers studying microbiology, antibiotic chemistry, β-lactamase enzymology. Thus, it is conceivable that scientists in diverse fields will be able to apply ML302 to elucidate the role of VIM-2 and IMP-1 in bacterial resistance pathways and investigate mechanisms of VIM-2 and IMP-1 inhibition in biochemical and microbiology-based assays.

Relevant biology of the probe. VIM-2 and IMP-1 are a zinc-dependent, Ambler Class “B” β-lactamases that hydrolyze β-lactam based antibiotics (e.g. penicillins, carbapenems), rendering them ineffective. No VIM-2 inhibitors yet exist for clinical use, and all VIM-2 inhibitors reported to date have been designed to bind zinc or modify cysteine found in the enzyme’s active site. Further, no dual-acting inhibitors exist which block activity of both of these enzymes. Therefore, selective, non-competitive VIM-2 inhibitors are desired to probe VIM-2 function exclusive of active site inhibition.

1. Introduction

The emergence of Gram-negative bacteria that exhibit multi-drug resistance, combined with the lack of new antibiotics, poses a public health challenge [3]. The production of bacterial β-lactamase enzymes, in particular, is a common mechanism of drug resistance [4–6]. The β-lactamases evolved from bacteria with resistance to naturally-occurring β-lactams or penams [7], agents which inhibit the transpeptidase involved in cell wall biosynthesis [8]. Human medicine adapted these agents into synthetic antibiotics such as penicillins, cephalosporins, carbapenems, and monobactams that contain a 2-azetidone ring [7, 9]. The metallo-β-lactamases (MBL) are zinc-dependent class B β-lactamases that hydrolyze the β-lactam ring, rendering the antibiotic ineffective [8, 10]. Increasingly, nosocomial beta-lactam antibiotic resistance arises in P. aeruginosa, Enterobacteriaceae, and other pathogenic bacteria via gene transfer of B1 MBLs [6, 11], including IMP (active on IMiPenem) [12] and VIM (Verona IMipenemase) [13, 14]. For two of these enzymes, VIM-2 and IMP-1, no inhibitors exist for clinical use [8, 11]. Thus, the identification of MBL inhibitors would provide useful tools for reducing nosocomial infections and elucidating their mechanism of action [2, 15]. In the present report, we identify and characterize a compound belonging to a novel rhodanine scaffold that inhibits both VIM-2 and IMP-1 with submicromolar Ki values. This represents a significant advance in the field of broad acting β-lactamase inhibitors.

2. Materials and Methods

2.1. Assays

Table 1Assays from the HTS campaign and prior probe discovery effort (ML121)

AIDAssay NameAssay TypeTargetPowder Sample
1527Primary biochemical HTS assay to identify inhibitors of VIM-2.Primary Assay (1X%INH)VIM-2No
1556Epi-absorbance primary biochemical HTS assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Primary Assay (1X%INH)IMP-1No
1856Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: biochemical HTS assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Counterscreen (3X%INH)IMP-1No
1857FRET-based counterscreen assay for selective VIM-2 inhibitors: biochemical HTS assay to identify epi-absorbance assay artifacts.Counterscreen (3X%INH)VIM-2 (CCF2)No
1860Epi-absorbance-based confirmation biochemical HTS assay to identify selective inhibitors of VIM-2 metallo-beta-lactamase.Confirmation (3X %INH)VIM-2No
1866Epi-absorbance-based counterscreen assay for selective VIM-2 inhibitors: biochemical HTS assay to identify inhibitors of TEM-1 serine-beta-lactamase.Counterscreen (3X%INH)TEM-1No
1919Epi-absorbance-based dose response biochemical high throughput screening assay for selective inhibitors of VIM-2 metallo-beta-lactamase.Dose Response (3X IC50)VIM-2No
1920Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Dose Response Counterscreen (3X IC50)IMP-1No
1925Epi-absorbance-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of TEM-1 serine-beta-lactamase.Dose Response Counterscreen (3X IC50)TEM-1No
1926FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify epi-absorbance assay artifacts.Dose Response Counterscreen (3X IC50)VIM-2 (CCF2)No
1927FRET-based counterscreen for selective VIM-2 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of IMP-1 metallo-beta-lactamase.Dose Response Counterscreen (3X IC50)IMP-1 (CCF2)No
1854Summary of probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase.SummaryVIM-2No
2128Late stage results from the probe development efforts to identify inhibitors of VIM-2: probe resultsLate Stage AID (probe)VIM-2Yes
2317Late stage results from the probe development efforts to identify selective inhibitors of VIM-2 metallo-beta-lactamase: Prior art results.Late Stage AID (prior art)VIM-2Yes
504620Late stage Assay provider assay to determine imipenem Synergy of probe ML121Late Stage AID (probe ML121)VIM-2Yes

Table 2Assays for the current probe discovery effort (ML302)

AIDAssay NameAssay TypeTargetPowder Sample
624079Absorbance-based Biochemical Nitrocefin Substrate Hydrolysis Assays (IC50)Dose Response (2X IC50) (probe ML302)VIM-2Yes
624085IMP-1Yes
624092TEM-1Yes
624090AmpCYes
624083Absorbance-based Biochemical Nitrocefin Substrate Hydrolysis Assays (Ki and Km)Dose Response (2X)VIM-2Yes
624084IMP-1Yes
624096Absorbance based Imipenem Antibiotic Synergy Assay (MIC4X): YMC07/B3323Dose Response (2X)VIM-2Yes
624081Absorbance based Imipenem Antibiotic Synergy Assay (MIC4X): BL21Dose Response (2X)VIM-2Yes
624080Absorbance based Imipenem Antibiotic Synergy Assay (MIC4X): PA641Dose Response (2X)VIM-2Yes
624082Absorbance based Imipenem Antibiotic Synergy Assay (MIC4X): BAA-2146Dose Response (2X)NDM-1Yes
624095Absorbance based Imipenem Antibiotic Synergy Assay (MIC4X): KN20Dose Response (2X)IMP-1Yes
624097Absorbance based Imipenem Antibiotic Synergy Assay (MIC4X): BL21Dose Response (2X)IMP-1Yes

(also see Summary AID 1854)

VIM-2 Inhibition Assays (Epi-Absorbance-based; Nitrocefin) (PubChem AIDs: AID 1527,AID 1860, AID 1919, AID 2128, AID 2317, and AID 624079). The purpose of this assay is to identify compounds that act as inhibitors of the VIM-2 β-lactamase. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the VIM-2 substrate, and takes advantage of the fluorescent properties of white microtiter plates [16]. Nitrocefin is a yellow chromogenic substrate (Imax = 395 nm) that is hydrolyzed by β-lactamases to yield a red product with increased absorbance properties (Imax = 495 nm) that quenches plate fluorescence by absorbing the plate’s emission light [16]. In this assay, test compounds are incubated with purified VIM-2 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit VIM-2 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds were tested in singlicate (AID 1527) and triplicate (AID 1860) at a final nominal concentration of 5.6 μM, in a 10-point 1:3 dilution series starting at a nominal concentration of 55.7 μM (AID 1919, AID 2128, AID 2317, and AID 624079).

IMP-1 Inhibition Counterscreens (Epi-absorbance-based; Nitrocefin) (AID 1556, AID 1856, AID 1920, AID 2128, AID 2317, and AID 624085). The purpose of this assay is to identify compounds that act as inhibitors of the IMP-1 β-lactamase. This biochemical epi-absorbance-format assay employs the cephalosporin nitrocefin as the IMP-1 substrate, and takes advantage of the fluorescent properties of white microtiter plates [16]. This assay also serves as a counterscreen to determine whether compounds identified as possible VIM-2 selective inhibitors are non-selective due to inhibition of IMP-1. Nitrocefin is a yellow chromogenic substrate (Imax = 395 nm) that is hydrolyzed by β-lactamases to yield a red product with increased absorbance properties (Imax = 495 nm) that quenches plate fluorescence by absorbing the plate’s emission light [16]. In this assay, test compounds are incubated with purified IMP-1 enzyme and nitrocefin in detergent-containing buffer at room temperature. The reaction is stopped by the addition of EDTA, followed by measurement of well fluorescence. As designed, compounds that inhibit IMP-1 will inhibit nitrocefin hydrolysis, inhibit generation of red product, and inhibit quenching of plate fluorescence, resulting in an increase in well fluorescence. Compounds were tested in singlicate (AID 1556) or in triplicate (AID 1856) at a final nominal concentration of 5.6 μM, and in triplicate using a dilution series starting at a nominal test concentration of 60 micromolar (AID 1920, AID 2128, AID 2317, and AID 624085).

2.2. Probe Chemical Characterization

The synthesis of the probe and analytical characterization data are probe ML302 are provided in Section 2.3. ML302 was obtained with >98% purity according to 1H NMR and LCMS analysis. However, ML302 had relatively poor solubility characteristics, so we also generated a more soluble HCl salt, identified as CID53384679. The HCl salt was also obtained with >96% purity by 1H NMR and LCMS analysis.

Solubility. The solubility of probe ML302 (synthesized and registered by the SRISMC as SR-03000002555-2/SID 134220672/CID 53362017) was measured in phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4) at room temperature (23°C). The solubility of was found to be <1 μM. In an attempt to achieve improved solubility, a series of salts (including the HCl salt, CID 53384679) were synthesized and studied. However, salt HCl CID 53384679 also had poor solubility (<1 μM) in this standard pH 7.4 PBS buffer assay system. However, probe ML302 and HCl salt CID53384679 are fully soluble at 100 μM in 70:30 DMSO/water, and both have solubility >100 μM at pH = 3 aqueous solution. A sample of the HCl salt CID 53384679 is also soluble >100 μM in D2O (1H NMR measurements performed at this concentration). Both probe ML302 and the HCl salt CID 53384679 are fully soluble under the conditions of the VIM-2 biochemical assays and antibacterial assays described in this probe report.

Stability. The stability of probe ML302 was measured at room temperature (23ºC) in PBS (no antioxidants or other protectants; DMSO concentration below 0.1%). The stability, represented by the half-life, was found to be very poor. Figure 1 provides graphs showing loss of compound with time over a 48 hour period with a minimum of 6 time points. The table at the end of this section indicates the percent of compound remaining at the end of the 48 hours. These data suggest that the probe ML302 and HCl salt CID 53384679 are highly unstable under these assay conditions. However, these data are not consistent with our experience with these compounds when handled under other conditions. Rather, we suspect that these data reflect solubility problems with the compound under the assay conditions. For example, HCl salt CID 53384679 proved to be fully stable, at 100 μM, in D2O over a two-week monitoring period (1H NMR study)—conditions under which it is fully soluble. Similarly, probe ML302 and HCl salt CID 53384679 were fully stable at 100 μM in 70/30 DMSO-D2O over a two-week period, in presence of air (1H NMR study)—conditions, again, under which they are fully soluble. ML302 and HCl salt CID 53384679 (both 1 μM) were fully stable (24 h monitoring) at pH 3 in PBS with 20% DMSO, and displayed >93% stability (over 24 h) at pH 7 PBS with 20% DMSO. The latter value undoubtedly reflects the limit of solubility of these compounds under these conditions. Apparently, the HCl salt CID 53384679 is converted to the free base (ML302) at pH 7.4.

Probe ML302, and its HCl salt were measured for its ability to form glutathione adducts. At concentrations of 100 μM reduced GSH, 10 μM of the new probe does not appear to be a Michael acceptor [17, 18]. Evidently, the chlorine substituents at the 2,6-position of the phenyl ring hinder the double bond such that these compounds are not highly active as Michael acceptors.

Table 3Solubility of ML302

CompoundMWSR NumberCIDSIDSolubility in PBS (μM)Michael Acceptor 100 μM GSH trapStability in PBS
t1/2 (hr)
New Probe ML302479.8SR-03000002555-253362017134220672<1 μMNoSection 2.2
Probe ML302
HCl salt		516.3SR-03000002594-253384679134220676 (synthesis)<1 μMNoSection 2.2

2.3. Probe Preparation

VIM-2 inhibitor probe ML302 was synthesized by a two-step procedure involving the condensation of 2,3,6-trichlorobenzaldehyde with rhodanine-3-acetic acid, followed by coupling of the product carboxylic acid 1 with 1-amino-4-methyl-piperazine [19].

2,3,6-Trichlorobenzaldehdye (1.676 g, 8.0 mmol, 1 equiv, purchased from Oakwood Product, Inc. # 043332) and rhodanine-3-acetic acid (1.530 g, 8.0 mmol, 1 equiv, purchased from Alfa Aesar, # B22244-06) were weighed into a 20-mL microwave vial. Ethanol (16 mL, to give a reaction concentration of 0.5 M) was added followed by piperidine (4 drops). The vial was sealed and submitted to microwave irradiation at 120 °C for 1 h. The solvent was then removed and the crude product was recrystallized from hot ethanol and water. The solid was filtered, rinsed with water and dried under high vacuum yielding compound 1 as a yellow solid (2.59 g, 85%): 1H NMR (400 MHz, d6-DMSO) δ 13.56 (1H, br s), 7.84 (1H, s), 7.87-7.84 (1H, m), 7.81 (1H, d, J = 8.8 Hz), 7.67 (1H, d, J = 8.7Hz), 4.70 (2H, s). 13C NMR (100 MHz, d6-DMSO) δ 192.6, 167.2, 164.9, 133.0, 132.2, 131.5, 131.4, 131.2, 131.1, 129.8, 128.6, 45.6.

A solution of 1 (76 mg, 0.2 mmol, 1 equiv.) and BOPCl (61 mg, 0.24 mmol, 1.2 equiv) in dichloromethane (2 mL, to give a reaction concentration of 0.1 M) in a 5-mL round-bottomed flask was cooled to 0 °C using an ice/water bath under an inert atmosphere. Freshly distilled triethylamine (35 μL, 0.24 mmol, 1.2 equiv) was added and the solution was allowed to stir at 0 °C for 30 min. The cold bath was removed and 1-amino-4-methyl-piperazine (29 μL, 0.24 mmol, 1.2 equiv, purchased from Acros Organics, # 251391000) was added. The mixture was allowed to warm to room temperature and was stirred overnight. The crude product, obtained by removal of all solvents volatile compounds in vacuo, was directly purified by flash chromatography (silica gel, dichloromethane-methanol 90/10, Rf = 0.29) yielding compound 2 as an inseparable mixture of E/Z isomers in a 1:3 ratio (75 mg, 78 %, yellow solid, mp = 184–186 °C) (Figure 2). The chemical purity of 2 is >98% according to HLPC analysis.

Data for VIM-2 Inhibitor Probe, CID 53362017 (Compound 2), Z isomer (major): 1H NMR (400 MHz, d6-DMSO) δ 9.08 (1H, s), 7.83 (1H, s), 7.82 (1H, d, J = 8.7 Hz), 7.68 (1H, d, J = 8.8 Hz), 4.91 (2H, s), 2.99-2.62 (6H, m), 2.43-2.02 (2H, m), 2.17 (3H, s); 13C NMR (100 MHz, d6-DMSO) δ 192.8, 166.0, 165.1, 133.0, 132.2, 131.5, 131.4, 131.3, 131.2, 129.8, 128.2, 55.1, 54.3, 45.3, 45.2. MS ([M+H]+): 481.1 (100%), 479.4 (71%), 483.0 (39%). IR (cm−1): 3049, 2927, 2808, 1731, 1687, 1627, 1402, 1366, 1323, 1286, 1273, 1193, 1175, 1051, 1008, 816, 725.

Partial Data for (minor) E isomer of VIM-2 Inhibitor Probe CID 53362017: 1H NMR (400 MHz, d6-DMSO) δ 9.37 (1H, s), 7.82 (1H, d, J = 8.7 Hz), 7.81 (1H, s), 7.68 (1H, d, J = 8.8 Hz), 4.56 (2H, s), 2.99-2.62 (6H, m), 2.43-2.02 (2H, m), 2.15 (3H, s).

All anti-bacterial and enzyme assays performed with the VIM-2 inhibitor probe, CID 53362017 (compound 2) were performed with the 3:1 mixture of olefin isomers. The two olefin isomers of the Probe were not separated, since prior studies with three probe analogs (SR-6818, SR-2448 and SR-2450) (Figure 3) indicated that the separated olefin isomers rapidly re-isomerized to the original 3:1 mixtures. (The olefin isomers of SR-6818, SR-2448 and SR-2450 were separated by preparative HPLC. In all three cases, the separated isomers had converted back to the original 3:1 mixtures in the time required to concentrate the HPLC fractions prior to NMR analysis).

A solution of hydrogen chloride in dioxane (4.0M, 4.1 mL, 16.4 mmol, 80 equiv) was slowly added at 0 °C to compound 2 (100 mg, 0.21 mmol, 1 equiv) under an inert atmosphere. The cold bath was removed and the mixture was allowed to stir at room temperature for 4 h. The resulting heterogeneous solution was filtered and the yellow precipitate 3 (Figure 4) was thoroughly washed with diethyl ether and dried under vacuum. No further purification was performed and the hydrochloride salt was obtained as a mixture of E/Z isomers in a 1:1 ratio (96 mg, 89 %, yellow solid, mp = 203–206 °C).

Data for VIM-2 Inhibitor CID 53384679 (Compound 3), Z isomer: 1H NMR (400 MHz, d6-DMSO) δ 11.21 (1H, br s), 9.41 (1H, s), 7.82 (1H, s), 7.81 (1H, d, J = 8.5 Hz), 7.67 (1H, d, J = 8.5 Hz), 4.99 (2H, s), 3.49-3.00 (8H, m), 2.73 (3H, s); 13C NMR (100 MHz, d6-DMSO) δ 192.8, 165.1, 162.1, 132.9, 132.2, 131.5, 131.4, 131.2, 131.1, 129.8, 128.3, 52.5, 51.8, 50.7, 45.3. MS ([M+H]+): 481.1 (100%), 479.5 (74%), 483.0 (38%). IR (cm−1): 2936, 2464, 1724, 1692, 1564, 1437, 1335, 1190, 1176, 1100, 1055, 984, 898, 814.

Partial Data for E isomer of VIM-2 Inhibitor Compound 3 CID 53384679: 1H NMR (400 MHz, d6-DMSO) δ 11.21 (1H, br s), 9.97 (1H, s), 7.81 (1H, d, J = 8.5 Hz), 7.80 (1H, s), 7.67 (1H, d, J = 8.5 Hz), 4.60 (2H, s), 3.49-3.00 (8H, m), 2.71 (3H, s).

3. Results

3.1. Summary of Screening Results

During the medicinal chemistry effort following our initial VIM-2 selective inhibitor project, the rhodanine scaffold, exemplified by SR-01000216818/CID1733268, exhibited activity against the anti-target enzyme, IMP-1. This suggested that this scaffold could be a source of broader-acting dual inhibitors of bacterial MBLs which might serve as tools for novel antibiotic discovery. In order to assess the efficacy of the new scaffold, we tested powder samples of leads in a series of biochemical and bacterial-based assays. Initial studies with recombinant VIM-2 protein revealed that several compounds in this scaffold exhibited potencies less than 5 micromolar. The broad acting MBL probe (ML302) was identified from this scaffold.

3.2. Dose Response Curves for Probes

Dose response curves for probe ML302 are shown in Figure 5 against all enzymes tested. The probe shows selective potency to the VIM-2 and IMP-1 enzymes. For comparison, SR-01000216818/CID 1733268 (“Analog 7”) is also graphed. Potency values are reported in the cover page of this report.

3.3. Scaffold/Moiety Chemical Liabilities

There is no known instability or chemical liabilities associated with the chemical scaffold of probe ML302. The compounds appear to be fully stable during all routine handling operations (synthesis chromatographic purification of the probe, NMR studies, storage as powders, etc.). Issues with solubility of the probe and its HCl salt are discussed in a previous section. The only issue we have observed is that the probe and its HCl salt are unstable at pH 10, due to amide hydrolysis at this basic pH. Therefore, handling of probe ML302 at pH > 7.4 is not recommended.

3.4. SAR Tables

Describe SAR & chemistry strategy (including structure and data) that led to the probe. Following submission of our initial probe report describing a selective VIM-2 inhibitor probe (ML121), compounds in the rhodanine scaffold appeared to have activity for both VIM-2 and IMP-1, suggesting a broad-acting MBL inhibitor probe could be developed. Powder and re-synthesized samples of selected compounds were tested by the assay provider in several biochemical and bacterial assays to determine potency, efficacy, and selectivity. These efforts resulted in probe ML302 and related analogs. The synthesis of ML302, presented in Section 2.3 of this probe report, is amenable to synthesis of additional analogs by substituting different aldehydes instead of 2,3,6-trichlorobenzaldehyde using the condensation of rhodanine-3-acetic acid; use of different amines instead of 1-amino-4-methyl-piperazine in the coupling with carboxylic acid 1. As summarized in the following SAR tables, more than 90 such analogs have been synthesized and tested. Preliminary efforts indicate that introduction of amino acid side chains in the rhodanine scaffold also lead to active compounds. Additional studies of such analogs will be performed in the future.

Metallo-beta Lactamase Inhibitor SAR Table: Rhodanine Scaffold

Compound InformationBioassaysBiochemical Assays
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-BAA-2146 (NDM-1)PA641 (VIM-2)KN20 (IMP-1)BL21 (IMP-1)VIM-2IMP-1TEM-1AmpCVIM-2 KiIMP-1 Ki Assay
MIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationIC50 (uM)IC50 (uM)IC50 (uM)IC50 (uM)Ki (nM)Km (uM)Ki (nM)Km (uM)
ProbeSR-03000002555-1
Image ml302fu2.jpg
53362017125264855TSRINone3.1258X0.78132X>502X504X>502X0.7810.54813.018>60>60NDNDNDND
Probe ML302 (Sample to NIH)SR-03000002555-2
Image ml302fu2.jpg
53362017134220672003940491TSRINoneND0.39132XNDNDNDNDNDNDNDND183 +/− 2414.8 +/− 3 Mixed mode inhibitor930 +/− 976.4 +/− 1.3 Mixed mode inhibitor
Analog 1SR-03000002674-1
Image ml302fu3.jpg
53495083126723255003940492TSRINone1.5630.78116X>50>50>503.125NDNDNDNDNDNDNDND
Analog 2SR-03000002704-1
Image ml302fu4.jpg
54579799131269026003940493TSRINone12.50.78132X>502X>502X>5025NDNDNDNDNDNDNDND
Analog 3SR-03000002705-1
Image ml302fu5.jpg
54579797131269027TSRINone6.251.56316X>50504X>500.7818XNDNDNDNDNDNDNDND
Analog 3SR-03000002705-2
Image ml302fu5.jpg
54579797134220673003940494TSRINoneND0.78132XNDNDNDNDNDNDNDNDNDNDNDND
Analog 4SR-03000002672-1
Image ml302fu6.jpg
53495082126723253TSRINone3.1251.56316X>50>50>500.781NDNDNDNDNDNDNDND
Analog 4SR-03000002672-2
Image ml302fu6.jpg
53495082134220674003940495TSRINoneND1.56332XNDNDNDNDNDNDNDNDNDNDNDND
Analog 5SR-03000002594-1
Image ml302fu7.jpg
53384679125311278TSRINone3.1258X1.56316X>50>50Achievs 2X>501.563NDNDNDNDNDNDNDND
Analog 5SR-03000002594-2
Image ml302fu7.jpg
53384679134220676003940496TSRINoneND0.78116XNDNDNDNDNDNDNDNDNDNDNDND
Compound InformationBioassaysBiochemical Assays
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-2)BAA-2146 (NDM-1)PA641 (VIM-2)KN20BL21VIM-2IMP-1VIM-2 Ki
MIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)MIC 4× (μM)IC50 (uM)IC50 (uM)Ki (nM)
Analog 6SR-03000002441-1
Image ml302fu8.jpg
53308605124767346TSRINone12.58X3.12532X>1002X>1002X>1003.125NDNDND
Analog 6 (resynthesized for submission to NIH)SR-03000002441-2
Image ml302fu8.jpg
53308605134220675003940497TSRINoneND3.12532XNDNDNDNDNDNDND
Analog 7 (Purchased)SR-01000216818-4
Image ml302fu9.jpg
1733268135631809ChemBridge56805644.551.781 +/− 0.384
Analog 7 (resynthesized)SR-01000216818-6
Image ml302fu9.jpg
1733268123083180TSRINoneND25Prepared in 75/25 DMSO/H2OND>100NDNDNDNDND
Analog 7 (purchased for reference)SR-01000216818-7
Image ml302fu9.jpg
1733268124360376ChemBridge5680564ND3.125Prepared in neat DMSONDNDNDNDNDNDND
Analog 7 (resynthesized)SR-01000216818-8
Image ml302fu9.jpg
1733268124384965003940498TSRINone6.258X6.2532X>100>1002X>100253.9831.9ND
Analog 8SR-03000002375-1
Image ml302fu10.jpg
53257035124398381TSRINone>100NDNDNDNDNDNDNDND
Analog 9SR-03000002376-1
Image ml302fu11.jpg
53257030124398382TSRINone>100NDNDNDNDNDNDNDND
Analog 10SR-03000002377-1
Image ml302fu12.jpg
53257026124398383TSRINone>100NDNDNDNDNDNDNDND
Compound InformationBioassays
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-2)BAA-2146 (NDM-1)PA641 (VIM-2)KN20BL21
MIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)MIC 4× (μM)
Analog 11SR-03000002380-1
Image ml302fu13.jpg
53257031124398386TSRINone>100NDNDNDNDND
Analog 12SR-03000002381-1
Image ml302fu14.jpg
53257037124398404TSRINone12.58XNDNDNDNDND
Analog 13SR-03000002461-1
Image ml302fu15.jpg
1382782125001880Vitas-M LabSTL035211ND6.2516XNDNDNDND
Analog 14SR-03000002378-1
Image ml302fu16.jpg
53257011124398384TSRINone6.28X6.2516X>100>1002X>100ND
Analog 15SR-03000002379-1
Image ml302fu17.jpg
53257036124398385TSRINone12.56.2516XNDNDNDND
Analog 16SR-03000002395-1
Image ml302fu18.jpg
53301882124756530TSRINone>100NDNDNDNDND
Analog 17SR-03000002396-1
Image ml302fu19.jpg
53301881124756531TSRINone>100NDNDNDNDND
Analog 18SR-03000002433-1
Image ml302fu20.jpg
53308614124767337TSRINone>100NDNDNDNDND
Analog 19SR-03000002434-1
Image ml302fu21.jpg
53308615124767338TSRINone>100NDNDNDNDND
Compound InformationBacterial Assays (Strain and MBL)Biochemical Assays
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-2)BAA-2146 (NDM-1)PA641 (VIM-2)KN20BL21VIM-2IMP-1VIM-2 Ki
MIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem PotentiationMIC 4× (μM)MIC 4× (μM)IC50 (uM)IC50 (uM)Ki (nM)
Analog 20SR-03000002455-1
Image ml302fu22.jpg
2049116125001873Innovapharm Ltd.STT-00175051>100NDNDNDNDNDNDNDND
Analog 21SR-03000002460-1
Image ml302fu23.jpg
5724963125001879Vitas-M LabSTL0352103.1350NDNDNDNDNDNDND
Analog 22SR-03000002385-1
Image ml302fu24.jpg
53301880124756519TSRINone25NDNDNDNDNDNDNDND
Analog 23SR-03000002386-1
Image ml302fu25.jpg
53301896124756520TSRINone>100NDNDNDNDNDNDNDND
Analog 24SR-03000002398-1
Image ml302fu26.jpg
53301889124756533TSRINone>100NDNDNDNDNDNDNDND
Analog 25SR-03000002447-1
Image ml302fu27.jpg
53313357124807342TSRINone>100NDNDNDNDNDNDNDND
Analog 26SR-03000002448-1
Image ml302fu28.jpg
53313358124807343TSRINone>100NDNDNDNDNDNDNDND
Analog 27SR-03000002457-1
Image ml302fu29.jpg
17592954125001875Vitas-M LabSTK626562100NDNDNDNDNDNDNDND
Analog 28SR-03000002459-1
Image ml302fu30.jpg
50739720125001877Innovapharm Ltd.STT-00362432100NDNDNDNDNDNDNDND
Analog 29SR-00000009750-1
Image ml302fu31.jpg
53308608124767339TSRINone>100NDNDNDNDNDNDNDND
Compound InformationBacterial Assays (Strain and MBL)
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-2)
MIC 4× (μM)Imipenem PotentiationMIC 4× (μM)Imipenem Potentiation
Analog 30SR-03000002382-1
Image ml302fu32.jpg
53257017124398405TSRINone12.58X6.2516X
Analog 31SR-03000002383-1
Image ml302fu33.jpg
53301893124756517TSRINone12.58XND
Analog 32SR-03000002432-1
Image ml302fu34.jpg
53308616124767336TSRINone>100ND
Analog 33SR-03000002374-1
Image ml302fu35.jpg
1560458124384967TSRINone12.58XND
Analog 34SR-03000002431-1
Image ml302fu36.jpg
53308606124767335TSRINone12.58XND
Analog 35SR-03000002442-1
Image ml302fu37.jpg
1280940124767347TSRINone100ND
Analog 36SR-03000002458-1
Image ml302fu38.jpg
1908865125001876Vitas-M LabSTK86177112.58XND
Analog 37SR-03000002397-1
Image ml302fu39.jpg
53301874124756532TSRINone>100ND
Analog 38SR-03000002454-1
Image ml302fu40.jpg
1554607125001872Innovapharm Ltd.STT-00172281>100ND
Analog 39SR-03000002456-1
Image ml302fu41.jpg
6226188125001874Vitas-M LabSTK601034>100ND
Compound InformationBacterial Assays (Strain and MBL)
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-2)BAA-2146 (NDM-1)PA641 (VIM-2)KN20
MIC 4x (μM)Imipenem PotentiationMIC 4x (μM)Imipenem PotentiationMIC 4x (μM)Imipenem PotentiationMIC 4x (μM)Imipenem PotentiationMIC 4x (μM)
Analog 40SR-03000002462-1
Image ml302fu42.jpg
5858470125001881Vitas-M LabSTL035212100NDNDNDND
Analog 41SR-03000002446-1
Image ml302fu43.jpg
53313356124807341TSRINone>100NDNDNDND
Analog 42SR-03000002451-1
Image ml302fu44.jpg
53313350124807346TSRINone>100NDNDNDND
Analog 43SR-03000002452-1
Image ml302fu45.jpg
53338906125001870TSRINone6.28X12.58X>100>100>100
Analog 44SR-03000002449-1
Image ml302fu46.jpg
53313352124807344TSRINone>100NDNDNDND
Analog 45SR-03000002450-1
Image ml302fu47.jpg
53313351124807345TSRINone>100NDNDNDND
Analog 46SR-03000002480-1
Image ml302fu48.jpg
53346521125011825TSRINone100NDNDNDND
Analog 47SR-03000002481-1
Image ml302fu49.jpg
53346520125011826TSRINoneND1.56332XNDNDND
Analog 48SR-03000002384-1
Image ml302fu50.jpg
53301887124756518TSRINone6.28X3.12516XND>100>100
Analog 49SR-03000002453-1
Image ml302fu51.jpg
53338912125001871TSRINone6.28X3.12516X>100>100>100
Analog 50SR-03000002482-1
Image ml302fu52.jpg
53346529125011827TSRINone3.1258X3.12516X>50>502X>50
Analog 51SR-03000002588-1
Image ml302fu53.jpg
53384710125311272TSRINone12.516X6.2532X>50ND>50
Analog 52SR-03000002589-1
Image ml302fu54.jpg
53384681125311273TSRINoneND3.125>50>50>50
Analog 53SR-03000002590-1
Image ml302fu55.jpg
53384726125311274TSRINoneND6.2532X>50>502X>50
Analog 54SR-03000002591-1
Image ml302fu56.jpg
53384676125311275TSRINoneND3.12516X>50>50>50
Analog 55SR-03000002592-1
Image ml302fu57.jpg
53384686125311276TSRINoneND25NDNDND
Analog 56SR-03000002593-1
Image ml302fu58.jpg
53384728125311277TSRINoneND>502XNDNDND
Analog 57SR-03000002737-1
Image ml302fu59.jpg
54669692131465571TSRINoneND258XNDNDND
Analog 58SR-03000002373-1
Image ml302fu60.jpg
1201205124384966TSRINone3.1258X12.516X>100>100>100
Analog 59SR-03000002519-1
Image ml302fu61.jpg
1205102125258648TSRINoneND>502XNDNDND
Compound InformationBacterial Assays (Strain and MBL)
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-2)
MIC 4x (μM)Imipenem PotentiationMIC 4x (μM)Imipenem Potentiation
Analog 60SR-03000002520-1
Image ml302fu62.jpg
1201114125258649TSRINoneND>502X
Analog 61SR-03000002521-1
Image ml302fu63.jpg
1622325125258651TSRINoneND>502X
Analog 62SR-03000002522-1
Image ml302fu64.jpg
13337884125258652TSRINoneND>502X
Analog 63SR-03000002523-1
Image ml302fu65.jpg
1201949125258653TSRINoneND>50
Analog 64SR-03000002524-1
Image ml302fu66.jpg
1201311125258654TSRINoneND>50
Analog 65SR-03000002525-1
Image ml302fu67.jpg
5943467125258655TSRINoneND>50
Analog 66SR-03000002526-1
Image ml302fu68.jpg
53356622125258656TSRINoneND>50
Analog 67SR-03000002527-1
Image ml302fu69.jpg
53356627125258657TSRINoneND>502X
Analog 68SR-03000002528-1
Image ml302fu70.jpg
53356652125258658TSRINoneND>502X
Analog 69SR-03000002529-1
Image ml302fu71.jpg
53356632125258660TSRINoneND6.2516X
Compound InformationBacterial Assays (Strain and MBL)
CompoundSR-NumberStructureCIDSIDMLSVendorVendor Catalog IDBL21 (VIM-2)YMC07/B3323 (VIM-2)BAA-2146 (NDM-1)PA641 (VIM-2)KN20
MIC 4x (μM)Imipenem PotentiationMIC 4x (μM)Imipenem PotentiationMIC 4x (μM)Imipenem PotentiationMIC 4x (μM)Imipenem PotentiationMIC 4x (μM)
Analog 70SR-03000002530-1
Image ml302fu72.jpg
53356630125258661TSRINoneND6.2516XNDNDND
Analog 71SR-03000002531-1
Image ml302fu73.jpg
53356624125258662TSRINoneND6.258XNDNDND
Analog 72SR-03000002586-1
Image ml302fu74.jpg
53384688125311270TSRINone258X252x>50ND>50
Analog 73SR-01000199849-2
Image ml302fu75.jpg
1381767125258650TSRINoneND>50NDNDND
Analog 74SR-01000214156-2
Image ml302fu76.jpg
1201186125258659TSRINoneND>502XNDNDND
Analog 75SR-01000686826-3
Image ml302fu77.jpg
1594367125001878Vitas-M LabSTK99403050NDNDNDND
Analog 76SR-01000699784-2
Image ml302fu78.jpg
1553748125001882Vitas-M LabSTL035275100NDNDNDND
Analog 77SR-03000002543-1
Image ml302fu79.jpg
53361995125264843TSRINoneND504XNDNDND
Analog 78SR-03000002544-1
Image ml302fu80.jpg
53361997125264844TSRINoneND>50NDNDND
Analog 79SR-03000002545-1
Image ml302fu81.jpg
53362020125264845TSRINoneND>50NDNDND
Analog 80SR-03000002546-1
Image ml302fu82.jpg
53362005125264846TSRINoneND>50NDNDND
Analog 81SR-03000002547-1
Image ml302fu83.jpg
53362003125264847TSRINoneND502XNDNDND
Analog 82SR-03000002548-1
Image ml302fu84.jpg
53362016125264848TSRINoneND254XNDNDND
Analog 83SR-03000002549-1
Image ml302fu85.jpg
53362008125264849TSRINoneND504XNDNDND
Analog 84SR-03000002550-1
Image ml302fu86.jpg
53362009125264850TSRINoneND>50NDNDND
Analog 85SR-03000002551-1
Image ml302fu87.jpg
53362004125264851TSRINoneND502XNDNDND
Analog 86SR-03000002552-1
Image ml302fu88.jpg
53362006125264852TSRINoneND502XNDNDND
Analog 87SR-03000002553-1
Image ml302fu89.jpg
53362014125264853TSRINoneND254XNDNDND
Analog 88SR-03000002554-1
Image ml302fu90.jpg
53362019125264854TSRINone12.53.12532X>502X>50>50
Analog 89SR-03000002556-1
Image ml302fu91.jpg
53362002125264856TSRINone1.5638X3.12532X>502X504X>50
Analog 90SR-03000002557-1
Image ml302fu92.jpg
53362007125264857TSRINone0.7818X3.12516X>502X>502X>50
Analog 91SR-03000002587-1
Image ml302fu93.jpg
53384696125311271TSRINone258X3.1252X>502X>50>50
Analog 92SR-03000002673-1
Image ml302fu94.jpg
53495084126723254TSRINoneND3.12516X>50>50>50
Analog 93SR-03000002706-1
Image ml302fu95.jpg
54579793131269028TSRINoneND6.2516XNDNDND
Analog 94SR-03000002707-1
Image ml302fu96.jpg
54579790131269029TSRINoneND>50NDNDND
Analog 95SR-03000002806-1
Image ml302fu97.jpg
56596523134228483TSRINoneND1.56332XNDNDND
Analog 96SR-03000002807-1
Image ml302fu98.jpg
56596515134228484TSRINoneND1.56332XNDNDND
Analog 97SR-03000002808-1
Image ml302fu99.jpg
56596526134228485TSRINoneND1.56332XNDNDND

3.5. Cellular Assays

The probe was tested in a variety of bacterial assays performed by the assay provider to determine the probe’s selectivity, efficacy and mechanism of action.

MIC and Imipenem Antibiotic Synergy Assays [AID 624081 BL21 (VIM2); AID 624097 BL21 (IMP1); AID 624096 YMC07 (VIM2); AID 624082 BAA2146 (NDM1); AID 624080 PA641 (VIM2); AID 624095 KN20 (IMP1)]. Using a checkerboard microdilution method, probe ML302 was tested for its ability to potentiate the efficacy of imipenem on VIM-2-expressing E. coli (BL21/VIM-2) [20–22]. The probe exhibited synergy with imipenem when present at concentrations as low as 3.13 μM, potentiating imipenem’s activity 8-fold. Significantly, probe ML302 exhibited 32-fold imipenem potentiation in clinically relevant VIM-2-transformed Acinetobacter species YMC07/B3323: MIC range = 390–781 nM). Synergy was also observed by probe ML302 in other clinical isolates including K. pneumonia BAA-2146 expressing NDM-1 [2-fold], P. aeruginosa expressing VIM-2 (PA641) [4-fold], and P. aeruginosa expressing IMP-1 (KN20) [2-fold].

3.6. Profiling Assays

The identified VIM-2/IMP-1 inhibitor probe ML302 was tested in biochemical and bacterial MIC assays to determine inhibition of VIM-2, IMP-1, TEM-1, AmpC, as well as assess its ability to potentiate the antibacterial activity of imipenem. Based on the results of these assays, it was agreed by the SRIMSC and assay provider that additional profiling was not required at this time.

4. Discussion

4.1. Comparison to existing art and how the new probe is an improvement

Compared to our prior probe ML121 and prior art mitoxantrone, the new probe ML302 exhibits improved biochemical potency, antibacterial activity, and IMP-1 activity.

Table 4Comparison of ML302 to existing Art

NameStructureSR NumberCIDSIDMLS IDVIM-2 IC50IMP-1 IC50VIM-2 KiTEM-1 IC50AmpC IC50SynergyPubChem Activity
Probe ML302
Image ml302fu1.jpg
SR-03000002555-253362017134220672MLS- 003940491548 nM3.02 μM183 nM>60 μM>60 μM3.125 μM (VIM-2)

0.781 μM (IMP-1)

(SID 125264855)		None
Probe ML121
Image ml302fu100.jpg
SR-01000775688-1487049424790728 (MLSMR) ;85856282 (purch); 103911139 (synth)MLS-000680027223 nM>60 μM148 nM>60 μMNDActive (12.5 μM) (TFA salt)6/309 (1.9%)
Prior art Mitoxantrone
Image ml302fu101.jpg
SR-01000076001-74212(5458171)56424031 (85856281)MLS 001333711powder 0.63 μM> 56 μM1.5 μM (non-competitive):> 56 μMNDNot applicable40/139 (28.8%)

4.2. Mechanism of Action Studies

The assay provider has performed assays to elucidate the mode of inhibition and the probe’s inhibitory constant (Ki) against VIM-2 and IMP-1. The results of these probe development efforts determined that probe ML302 is a non-selective, mixed mode inhibitor probe of the metallo-beta-lactamases VIM-2 and IMP-1 (Figure 6).

VIM-2 and IMP-1 Ki Assays (AID 624083 andAID 624084). The purpose of these assays is to determine the inhibition constant (Ki) and modality of probe candidate molecules. Kinetic assays were conducted by incubating a range of nitrocefin substrate concentrations (100nM - 5 μM) with varying inhibitor concentrations and 0.1 nM enzyme at room temperature in buffer containing 50mM HEPES, 50μM ZnSO4, 0.05% Brij 35, pH 7.1. Absorbance was measured on a Tecan Safire2 monochromatic microplate reader at 495 nm. Initial velocities were obtained from plots of absorbance at 495 nm versus time, using data points from only the linear portion of the hydrolysis curve. Substrate hydrolysis was continuously monitored. Initial velocities were plotted vs. substrate concentration and kinetic parameters were calculated using Graphpad Prism version 5.01 suite of programs. Mode of inhibition was determined using fit comparison capability of Graphpad Prism version 5.01 and additionally evaluated by Lineweaver-Burke plot. Ki values were determined by non-linear regression analysis. The results of these studies demonstrated that probe ML302 (CID 53362017) is a mixed mode uncompetitive and non-competitive inhibitor, with a submicromolar VIM-2 (183 ± 24 nM) and IMP-1 (930 ± 97 nM) Ki values (Figure 6).

Table 5Ki values demonstrating ML302 is a mixed mode uncompetitive and non-competitive inhibitor

ProbeCorp IDSIDCIDModalityVim-2 Ki, nMIMP-1 Ki, nM
New Probe ML302SR-03000002555-213422067253362017Mixed mode Inhibitor183930
Prior Probe ML121SR-01000775688-3858562824870494Non-competitive inhibition148ND

4.3. Planned Future Studies

The assay provider plans to determine the efficacy of probe ML302 as well as prior probe ML121 in eukaryotic cells and in animal models of bacterial infection. Additional studies will explore the SAR around the rhodanine scaffold.

5. References

1.
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Minond D, Saldanha SA, Subramaniam P, Spaargaren M, Spicer T, Fotsing JR, Weide T, Fokin VV, Sharpless KB, Galleni M, Bebrone C, Lassaux P, Hodder P. Inhibitors of VIM-2 by screening pharmacologically active and click-chemistry compound libraries. Bioorg Med Chem. 2009;17(14):5027–37. [PMC free article: PMC2759276] [PubMed: 19553129]
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Siegel RE. Emerging gram-negative antibiotic resistance: daunting challenges, declining sensitivities, and dire consequences. Respir Care. 2008;53(4):471–9. [PubMed: 18364060]
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Gupta V. An update on newer beta-lactamases. Indian J Med Res. 2007;126(5):417–27. [PubMed: 18160745]
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Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001;14(4):933–51. table of contents. [PMC free article: PMC89009] [PubMed: 11585791]
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Sacha P, Wieczorek P, Hauschild T, Zorawski M, Olszanska D, Tryniszewska E. Metallo-beta-lactamases of Pseudomonas aeruginosa--a novel mechanism resistance to beta-lactam antibiotics. Folia Histochem Cytobiol. 2008;46(2):137–42. [PubMed: 18519228]
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Koch AL. Bacterial wall as target for attack: past, present, and future research. Clin Microbiol Rev. 2003;16(4):673–87. [PMC free article: PMC207114] [PubMed: 14557293]
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Jin W, Arakawa Y, Yasuzawa H, Taki T, Hashiguchi R, Mitsutani K, Shoga A, Yamaguchi Y, Kurosaki H, Shibata N, Ohta M, Goto M. Comparative study of the inhibition of metallo-beta-lactamases (IMP-1 and VIM-2) by thiol compounds that contain a hydrophobic group. Biol Pharm Bull. 2004;27(6):851–6. [PubMed: 15187432]
9.
Abeylath SC, Turos E. Drug delivery approaches to overcome bacterial resistance to beta-lactam antibiotics. Expert Opin Drug Deliv. 2008;5(9):931–49. [PubMed: 18754746]
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Wang Z, Fast W, Valentine AM, Benkovic SJ. Metallo-beta-lactamase: structure and mechanism. Curr Opin Chem Biol. 1999;3(5):614–22. [PubMed: 10508665]
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Walsh TR, Toleman MA, Poirel L, Nordmann P. Metallo-beta-lactamases: the quiet before the storm? Clin Microbiol Rev. 2005;18(2):306–25. [PMC free article: PMC1082798] [PubMed: 15831827]
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Hirakata Y, Izumikawa K, Yamaguchi T, Takemura H, Tanaka H, Yoshida R, Matsuda J, Nakano M, Tomono K, Maesaki S, Kaku M, Yamada Y, Kamihira S, Kohno S. Rapid detection and evaluation of clinical characteristics of emerging multiple-drug-resistant gram-negative rods carrying the metallo-beta-lactamase gene blaIMP. Antimicrob Agents Chemother. 1998;42(8):2006–11. [PMC free article: PMC105724] [PubMed: 9687398]
13.
Lauretti L, Riccio ML, Mazzariol A, Cornaglia G, Amicosante G, Fontana R, Rossolini GM. Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob Agents Chemother. 1999;43(7):1584–90. [PMC free article: PMC89328] [PubMed: 10390207]
14.
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Figures

Figure 1. Stability Analysis of ML302 and its HCl salt.

Figure 1Stability Analysis of ML302 and its HCl salt

Figure 2. Synthesis of VIM-2 Inhibitor Probe CID 53362017 (ML302) (Compound 2).

Figure 2Synthesis of VIM-2 Inhibitor Probe CID 53362017 (ML302) (Compound 2)

Figure 3. Probe analogs.

Figure 3Probe analogs

Figure 4. Synthesis of Hydrochloride Salt of the Probe (CID 53384679, Compound 3).

Figure 4Synthesis of Hydrochloride Salt of the Probe (CID 53384679, Compound 3)

Figure 5. Dose response curves for ML302.

Figure 5Dose response curves for ML302

Figure 6. Mixed mode of inhibition for VIM-2 and IMP-1.

Figure 6Mixed mode of inhibition for VIM-2 and IMP-1